AU2016349392A1

AU2016349392A1 - Neutralizing antibodies to HIV-1 gp41 and their use

Info

Publication number: AU2016349392A1
Application number: AU2016349392A
Authority: AU
Inventors: Mangaiarkarasi ASOKAN; Robert BAILER; Michael Bender; Kevin Carlton; Gwo-yu CHUANG; Mark Connors; Jonathan Cooper; Ivelin GEORGIEV; Tatyana GINDIN; Lisa KUELTZO; Young Do Kwon; Peter Kwong; Mark LOUDER; John MACSOLA; Krisha MCKEE; Sijy O'DELL; Gilad Ofek; Amarendra PEGU; Richard Schwartz; Lawrence Shapiro
Original assignee: Columbia University in the City of New York; Government of the United States of America
Current assignee: Columbia University in the City of New York; US Department of Health and Human Services
Priority date: 2015-11-03
Filing date: 2016-11-03
Publication date: 2018-05-24
Anticipated expiration: 2036-11-03
Also published as: AU2016349392B2; US11236152B2; WO2017079479A1; CN108884149B; CN115010805B; EP4011911A1; CN108884149A; CA3003878A1; US12043660B2; US20190077849A1; WO2017079479A8; CN115010805A; US20220098286A1; EP3371214A1

Abstract

Neutralizing antibodies that specifically bind to HIV-1 Env and antigen binding fragments of these antibodies are disclosed. Nucleic acids encoding these antibodies, vectors and host cells are also provided. Methods for detecting HIV-1 using these antibodies are disclosed. In addition, the use of these antibodies, antigen binding fragment, nucleic acids and vectors to prevent and/or treat an HIV-1 infection is disclosed.

Description

BACKGROUND

HIV-1 infection, and the resulting Acquired Immunodeficiency Syndrome (AIDS), remain threats to global public health, despite extensive efforts to develop anti-HIV-1 therapeutic agents. An enveloped virus, HIV-1 hides from humoral recognition behind a wide array of protective mechanisms. The major HIV-1 envelope protein (HIV-1 Env) is a glycoprotein of approximately 160 kD (gpl60). During infection, proteases of the host cell cleave gpl60 into gpl20 and gp41. gp41 is an integral membrane protein, while gpl20 protrudes from the mature virus. Together gpl20 and gp41 make up the HIV-1 envelope spike, which is a target for neutralizing antibodies. Broadly neutralizing antibodies that bind to HIV-1 Env have been identified, including the 10E8 antibody, which specifically binds to the membrane proximal external region (MPER) of gp41 and can neutralize a high percentage of HIV-1 strains. However, there is a need to develop additional neutralizing antibodies for HIV-1 with varying recognition and neutralization profiles for commercial production.

SUMMARY

Disclosed herein are isolated antibodies and antigen binding fragments that specifically bind to HIV1 gp41 and neutralize HIV-1. In some embodiments, the isolated antibody or antigen binding fragment comprises heavy and light chain variable regions comprising one or more amino acid substitutions (for example in the complementarity determining regions (CDRs) and/or the framework regions) compared to the 10E8 antibody sequence that impart an improved combination of neutralization, solubility, and autoreactivity properties relative to the 10E8 antibody.

In some embodiments, the isolated antibody or antigen binding fragment comprises a heavy chain variable region (Vh) comprising a heavy chain complementarity determining region (HCDR)l, a HCDR2, and a HCDR3 of the Vh set forth as SEQ ID NO: 75 (10E8v4 SlOOcF), and a light chain variable region (V_L) comprising a light chain complementarity determining region (FCDR)l, a FCDR2, and a FCDR3 of the Vl set forth as SEQ ID NO: 6 (rF3-6mut), wherein the antibody or antigen binding fragment specifically binds to gp41 and neutralizes HIV-1. In some embodiments, the HCDR1, the HCDR2, the HCDR3, the

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LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 7, 8, 77,

10, 11, and 12, respectively (10E8v4 SlOOcF IMGT CDRs). In some embodiments, the Vh and Vl of the isolated antibody or antigen binding fragment comprise the amino acid sequences set forth as SEQ ID NOs:

and 6, respectively (10E8v4 SlOOcF), or SEQ ID NOs: 76 and 6, respectively (10E8v4 V5R SlOOcF), wherein the antibody or antigen binding fragment specifically binds to gp41 and neutralizes HIV-1.

In additional embodiments, the isolated antibody comprises a Vh comprising a HCDR1, a HCDR2, and a HCDR3 of the Vh set forth as SEQ ID NO: 5, and a Vl comprising a light chain LCDR1, a LCDR2, and a LCDR3 of the Vl set forth as SEQ ID NO: 6, wherein the Vh comprises arginine, glutamate, serine, lysine, aspartate, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively, and the Vl comprises alanine, serine, aspartate, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, and wherein the antibody or antigen binding fragment specifically binds to gp41 and neutralizes HIV-1.

Also disclosed are compositions including the antibodies and antigen binding fragments, nucleic acids encoding the antibodies and antigen binding fragments, expression vectors comprising the nucleic acids, and isolated host cells that comprise the nucleic acids. In some embodiments, the nucleic acid molecule can be a bicistronic expression construct encoding the Vh and Vl of the antibody or antigen binding fragment.

The disclosed antibodies and antigen binding fragments potently neutralize HIV-1 in an accepted in vitro model of HIV-1 infection. Accordingly, a method is disclosed for treating or inhibiting an HIV-1 infection in a subject. The methods include administering a therapeutically effective amount of one or more of the disclosed antibodies, antigen binding fragments, nucleic acid molecules, vectors, or compositions, to the subject, for example to a subject at risk of or having an HIV-1 infection.

The antibodies, antigen binding fragments, nucleic acid molecules, vectors, and compositions disclosed herein can be used for a variety of additional purposes, such as for detecting an HIV-1 infection or diagnosing HIV-1 infection in a subject, or detecting HIV-1 in a sample.

The foregoing and other features and advantages of this disclosure will become more apparent from the following detailed description of several embodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGs. 1A-1F are sequence alignments of antibody heavy and light chain variable domains. FIGs.

1A and IB show kabat numbering and IMGT (FIG. 1 A) or kabat (FIG. IB) CDR positioning of the 10E8 (SEQ ID NO: 1), HC6-S74Y (SEQ ID NO: 13), HC6-S74Y-511 (SEQ ID NO: 3), H6 (SEQ ID NO: 14),

H6-511 (SEQ ID NO: 15), and H6-511-4mut (SEQ ID NO: 5) heavy chain variable domains. FIGs. 1C and

ID show kabat numbering and IMGT (FIG. 1C) or kabat (FIG. ID) CDR positioning of the 10E8 (SEQ ID

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NO: 2), rL3 (SEQ ID NO: 4), and rL3-6mut (SEQ ID NO: 6) light chain variable domains. FIG. IE shows kabat numbering and IMGT CDR positioning of the V_H for 10E8 (SEQ ID NO: 1), 10E8v4 (SEQ ID NO: 5),

10E8v4 SlOOcF (SEQ ID NO: 75), and 10E8v4 V5R SlOOcF (SEQ IDNO: 76).FIG. IF shows kabat numbering and IMGT CDR positioning of the V_L for 10E8 (SEQ ID NO: 2) and 10E8v4 (SEQ ID NO: 6), which is also used at the VL for 10E8v4 SlOOcF and 10E8v4 V5R SlOOcF.

FIG. 2 is a graph and an image of a coommassie blue stained gel illustrating the turbidity (as measured at OD350) in phosphate buffered saline of the 10E8 antibody and 10E8 variants including the HC6-S74Y V_H and the rL3 V_L, or the HC6-S74Y-511 V_H and the rL3 V_L. The highly soluble VRC01 antibody was used as a control.

FIG.3 is a table illustrating neutralization of a representative panel of HIV-1 pseudoviruses by the 10E8 antibody, and 10E8 variants including the HC6-S74Y Vh and the rL3 Vl, or the HC6-S74Y-511 Vh and the rL3 Vl. Neutralization was determined using the TZM-bl cell pseudovirus neutralization assay; ICso values are shown.

FIG. 4A is a graph illustrating the turbidity (as measured at OD350) in phosphate buffered saline of the 10E8 antibody and 10E8 variants including the H6 Vh and the L10 Vl, the H8 Vh and the L10 Vl, the H8 Vh and the L19 Vl, the H6-511 Vh and the L10 VL.or the H8-511 Vh and the L10 Vl. The highly soluble VRC01 antibody was used as a control.

FIG. 4B is a table illustrating neutralization of a representative panel of HIV-1 pseudo viruses by the 10E8 antibody, and 10E8 variants including the H6-511 Vh and the L10 Vl, or the H8-511 Vh and the L10 Vl. Neutralization was determined using the TZM-bl cell pseudovirus neutralization assay; IC50 values are shown.

FIG. 5 is a ribbon diagram illustrating a portion of the structure of the 10E8 antibody bound to the MPER peptide. The “4mut” residues D28, N31, T52, and Y98 (kabat numbering) of the heavy chain variable domain are highlighted.

FIG. 6 is a graph illustrating the turbidity (as measured at OD350) in phosphate buffered saline of the 10E8 antibody and the indicated 10E8 variants. The highly soluble VRC01 antibody was used as a control.

FIG. 7 is a table illustrating neutralization of a representative panel of HIV-1 pseudoviruses by the 10E8 antibody, and 10E8 variants including the H6-511 Vh and the rL3 Vl, or the H6-511 -4mut (H6-511 with N28D, D31N, S52T, and H98Y substitutions) Vh and the L10 Vl. Neutralization was determined using the TZM-bl cell pseudovirus neutralization assay; IC50 values are shown.

FIG. 8 is a graph illustrating the turbidity (as measured at OD350) in phosphate buffered saline of the 10E8 antibody and the 10E8 variants including the HC6-S74Y-511 Vh and the L10 Vl, the HC6-S74Y Vh and the L10 Vl, or the 10E8 Vh and the L10 Vl. The highly soluble VRC01 antibody was used as a control.

FIG. 9 is a graph illustrating the turbidity (as measured at OD350) in phosphate buffered saline of the 10E8 antibody and 10E8 variants including the 10E8 Vh paired with the indicated Vl domains. The rL3

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V_L with SI A, Y2S, T8P, G9A, G16K, R17Q substitutions (“rL3-6mut”) paired with the 10E8 V_H had the least turbidity of the 10E8 variants. The highly soluble VRC01 antibody was used as a control.

FIG. 10 is a graph illustrating the turbidity (as measured at OD350) in phosphate buffered saline of the 10E8 antibody and the 10E8 variants including the H6-511-4mut Vh and the rL3 Vl, or the H6-511-4mut

Vh and the rL3-6mut Vl. The highly soluble VRC01 antibody was used as a control.

FIG. 11 is a table illustrating neutralization of a representative panel of HIV-1 pseudo viruses by the 10E8 antibody, and the 10E8 variants including the H6-511 Vh and rL3 Vl, the H6-511-4mut Vh and rL3, or the H6-51 l-4mut Vh and the rL3-6mut Vl. Neutralization was determined using the TZM-bl cell pseudovirus neutralization assay; IC50 and IC₈o values are shown.

FIG. 12 is a flowchart illustrating the development of modified 10E8 antibodies.

FIGs. 13A-13C illustrate the results of kinetic concentration assays of the 10E8 antibody or the 10E8 variants including the HC6-S74Y-511 Vh and the rL3 Vl, the HC6-S74Y-511 Vh and the rL3, or the H6-511-4mut and the rL3-6mut Vl. Three milliliters of the indicated antibodies (OD 0.35) were concentrated using a centrifugal concentrator (FIG. 13 A) and the volume (FIG. 13B) and protein concentration (FIG. 13C, OD280) of resulting concentrate was measured.

FIG. 14 is a set of graphs showing the results of dynamic light scattering assays of solutions containing the 10E8 antibody or a 10E8 variant including the HC6-S74Y-511 Vh and the rL3 Vl, the HC6S74Y-511 Vh and the rL3, or the H6-511 -4mut and the rL3-6mut Vl. The 10E8 antibody was polydisperse, indicating the presence of protein aggregates. Antibodies including the HC6-S74Y-511 Vh and the rL3 Vl, the HC6-S74Y-511 Vh and the rL3, or the H6-511-4mut and the rL3-6mut Vl were monodisperse.

FIG. 15 is a set of immunofluorescence images illustrating that the 10E8 antibody and the 10E8 variants including the HC6-S74Y-511 Vh and the rL3 Vl, the HC6-S74Y-511 Vh and the rL3-6mut Vl, or the H6-51 l-4mut and the rL3-6mut Vl are not autoreactive. Reactivity to HIV-1 negative human epithelial (HEp-2) cells was determined by indirect immunofluorescence.

FIGs. 16A-16F are a set of tables illustrating the neutralization of a panel of 200 HIV-1 pseudoviruses by the 10E8 antibody and 10E8 variants including the HC6-S74Y-511 Vh and the rL3 Vl (Varl), the HC6-S74Y-511 Vh and the rL3-6mut Vl (Var5), or the H6-511-4mut and the rL3-6mut Vl (Var4). The IC50 (FIGs. 16A-16C) and IC₈₀ (FIGs. 16D-16F) values are shown.

FIG. 17 is a graph showing the concentration of the 10E8 antibody or 10E8 variant antibody in primate (macaque) serum over time following a single injection of antibody.

FIGs. 18A-18D are a set of graphs illustrating the size exclusion chromatography of 10E8, 10E8v4, and 10E8v5 (FIG. 18A), size exclusion chromatography of several 10E8v4 variants with amino acid substitutions at Vl kabat position 32 (FIGs. 18B and 18C), and the turbidity of several 10E8v4 variants with amino acid substitutions at Vl kabat position 32 (FIGs. 18D).

FIG. 19 shows a set of tables illustrating IC50 and IC₈o values for the neutralization of a panel of

HIV-1 viruses by several broadly neutralizing HIV-1 antibodies, including the 10E8v4 SlOOcW antibody.

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FIG. 20 is a set of graphs and ribbon diagrams illustrating that alteration of the 10E8v4 HCDR3 can refine the size exclusion chromatography profile to a single peak.

FIG. 21 is a set of graphs showing size exclusion chromatography results for the 10E8v4-DS and

10E8v4-100cW-DS antibodies.

FIG. 22 is an illustration of the positioning within the Cryo-EM HIV-1 Env structure fitted with the 10E8v4 antibody of certain amino acid substitutions to 10E8v4 that improve HIV-1 neutralization. One group of mutations was in a membrane-proximal region of 10E8v4 when 10E8v4 is bound to HIV-1 Env (D7W, D26R, and G68R substitutions to the Vl), and another group was in a region of 10E8v4 that is not proximal to the cell membrane (D72R, E1R D28W, and P52bR substitutions to the Vh).

FIGs. 23A-23F show a set of tables illustrating neutralization of a representative panel of HIV-1 pseudoviruses by several different 10E8 variant antibodies. The 10E8 variants included those in Table 10 (see Example 5). Neutralization was determined using the TZM-bl cell pseudovirus neutralization assay;

IC₅₀ (FIGs. 23A, 23C, and 23D) and IC₈₀ (FIGs. 23B, 23E, 23F) values are shown.

FIG. 24 is a set of graphs showing results from solubility assays of 10E8v4 variants including SlOOcF, SlOOcH, SlOOcL, SlOOcR, SlOOcW, or SlOOcY substitutions performed by measuring the volume and protein concentration of solutions of these antibodies following concentration using a standard centrifugal concentrator.

SEQUENCES

The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file in the form of the file named “Sequence.txt” (-165 kb), which was created on November 1, 2016, and which is incorporated by reference herein. In the accompanying sequence listing:

SEQ ID NO: 1 is the amino acid sequence of the 10E8 Vh.

EVQLVESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAAPVEGRFTISRLNSINF

LYLEMNNLRMEDSGLYFCARTGKYYDFWSGYPPGEEYFQDWGRGTLVTVSS

SEQ ID NO: 2 is the amino acid sequence of the 10E8 Vl.

SYELTQETGVSVALGRTVTITCRGDSLRSHYASWYQKKPGQAPILLFYGKNNRPSGVPDRFSGSASGNRASLTISGAQAE

DDAEYYCSSRDKSGSRLSVFGGGTKLTVL

SEQ ID NO: 3 is the amino acid sequence of the HC6-S74Y-511 Vh.

EVRLAESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAAPVEGRFTISRDNYKNT

LYLEMNNLRTEDSGLYFCARTGKYYDFWSGYPPGEEYFQDWGRGTLVIVSS

SEQ ID NO: 4 is the amino acid sequence of the rL3 Vl.

SYELTQDTGVSVALGRTVTITCRGDSLRSHYASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSGSASGNRASLTITGAQAE

DEADYYCSSRDKSGSRLSVFGGGTKLTVL

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SEQ ID NO: 5 is the amino acid sequence of the H6-51 l-4mut Vh.

EVRLVESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAESVKGRFTISRDNTKNT

LYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGEEYFQDWGQGTLVIVSS

SEQ ID NO: 6 is the amino acid sequence of the rL3-6mut VlASELTQDPAVSVALKQTVTITCRGDSLRSHYASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSGSASGNRASLTITGAQAE

DEADYYCSSRDKSGSRLSVFGGGTKLTVL

SEQ ID NOs: 7-12 are amino acid sequences of the IMGT CDRs of the 10E8 antibody.

SEQ ID NO: 13 is the amino acid sequence of the HC6-S74Y Vh.

EVRLAESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAAPVEGRFTISRLNYINF

LYLEMNNLRMEDSGLYFCARTGKYYDFWSGYPPGEEYFQDWGRGTLVIVSS

SEQ ID NO: 14 is the amino acid sequence of the H6 Vh.

EVRLVESGGGLVKPGGSLRLSCSASGFNFDDAWMTWVRQPPGKGLEWVGRISGPGEGWSVDYAESVKGRFTISRLNSINF

LYLEMNNVRTEDTGYYFCARTGKHYDFWSGYPPGEEYFQDWGQGTLVIVSS

SEQ ID NO: 15 is the amino acid sequence of the H6-511 Vh.

EVRLVESGGGLVKPGGSLRLSCSASGFNFDDAWMTWVRQPPGKGLEWVGRISGPGEGWSVDYAESVKGRFTISRDNTKNT

LYLEMNNVRTEDTGYYFCARTGKHYDFWSGYPPGEEYFQDWGQGTLVIVSS

SEQ ID NO: 16 is the amino acid sequence of the H8 Vh.

EVRLVESGGRLVRPGGSLRLSCSASGFNFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAASVKGRFTISRMNSINF

FYLEMNNLKIEDTGLYFCARTGKHYAFWGGYPPGEEYLEDWGQGTLVIVSS

SEQ ID NO: 17 is the amino acid sequence of the H8-511 Vh.

EVRLVESGGRLVRPGGSLRLSCSASGFNFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAASVKGRFTISRDNYKNT

FYLEMNNLKTEDTGLYFCARTGKHYAFWGGYPPGEEYLEDWGQGTLVIVSS

SEQ ID NO: 18 is the amino acid sequence of the L10 Vl.

ASELTQDPAVSVALKQTVTITCRGDSLRSHYVSWYQKKPGQAPVLVFYGKNNRPSGIPDRFSGSSSGNTASLTIAGAQAE

DDADYYCSSRDKSGSRLSVFGGGTKLTVL

SEQ ID NO: 19 is the amino acid sequence of the L19 Vl.

ASELTQDPTVSVALGQTVTITCRGDSLRNYYTSWYQQKPGQAPVLLIYPKHNRPPGISDRFSASSSGNTASLTITGAQTE

DEGDYYCSSRDKSGSRLVTFGRGTKLTW

SEQ ID NO: 20 is an exemplary nucleic acid sequence encoding the HC6-S74Y-511 Vh linked to an IgGl constant region.

atgggatggtcatgtatcatcctttttctagtagcaactgcaaccggtgtacattctgaagtgcggctggctgagagcgg cggggggctggtcaaacctggcgggtcactgcggctgtcctgttctgcctccggcttcgattttgataacgcatggatga catgggtgcgacagccacctggaaaggggctggagtgggtcggcagaatcactggacctggcgaagggtggtctgtggac tacgcagctccagtcgagggacgattcaccattagtagagataactacaagaatacactgtatctggagatgaacaatct gaggactgaagacagcggcctgtatttctgcgcccgcaccgggaaatactatgatttttggtctgggtacccacccggag aggaatattttcaggactggggacggggcaccctggtgatcgtcagctccgcgtcgaccaagggcccatcggtcttcccc ctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggt gacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctact ccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagc aacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaact cctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacat gcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgcc aagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggct gaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaag

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PCT/US2016/060390 ggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgc ctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccac gcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcagggga acgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa tga

SEQ ID NO: 21 is an exemplary nucleic acid sequence encoding the H6-51 l-4mut Vh linked to an IgGl constant region.

atgggatggtcatgtatcatcctttttctagtagcaactgcaaccggtgtacattctgaggttagactggtggagtcagg aggggggcttgtgaagcccggtgggtctctccgcctgagctgttctgcctccggctttgatttcgataacgcctggatga cctgggtcaggcagcctccaggtaagggactggagtgggtgggaagaatcacaggtccaggcgagggctggtccgtggac tacgcggaatctgttaaagggcggtttacaatctcaagggacaataccaagaataccttgtatttggagatgaacaacgt gagaactgaagacaccggatattacttctgtgccagaacaggcaaatactacgacttctggtccggctatccccctggcg aggaatattttcaagactggggtcagggaacccttgttatcgtgtcctccgcgtcgaccaagggcccatcggtcttcccc ctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggt gacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctact ccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagc aacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaact cctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacat gcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgcc aagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggct gaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaag ggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgc ctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccac gcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcagggga acgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa

SEQ ID NO: 22 is the amino acid sequence of the H6-51 l-4mut Vh linked to an IgGl constant region.

EVRLVESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAESVKGRFTI SRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGEEYFQDWGQGTLVIVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 23 is the amino acid sequence of the HC6-S74Y-511 Vh linked to an IgGl constant region.

EVRLAESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAAPVEGRFTI SRDNYKNTLYLEMNNLRTEDSGLYFCARTGKYYDFWSGYPPGEEYFQDWGRGTLVIVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 24 is an exemplary nucleic acid sequence encoding the rL3 Vl.

tcatacgaactgactcaggacactggcgtctctgtggcactggggaggactgtgactattacttgccgaggcgactcact gcggagccactacgcttcctggtatcagaagaaacccggccaggcacctgtgctgctgttctacggaaagaacaataggc catctggcatccccgaccgcttttctggcagtgcatcagggaaccgagccagtctgaccattaccggcgcccaggctgag gacgaagccgattactattgcagctcccgggataagagcggctccagactgagcgtgttcggaggaggaactaaactgac cgtcctcagtcagcccaaggctgccccctcggtcactctgttcccgccctcgagtgaggagcttcaagccaacaaggcca cactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcg ggagtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctacctgagcctgacgcctgagca

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PCT/US2016/060390 gtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaat gttcatag

SEQ ID NO: 25 is an exemplary nucleic acid sequence encoding the rL3-6mut Vlaccaccatgggatggtcatgtatcatcctttttctagtagcaactgcaaccggttctgtgaccgcatccgaactgactca ggaccctgccgtctctgtggcactgaagcagactgtgactattacttgccgaggcgactcactgcggagccactacgctt cctggtatcagaagaaacccggccaggcacctgtgctgctgttctacggaaagaacaataggccatctggcatccccgac cgcttttctggcagtgcatcagggaaccgagccagtctgaccattaccggcgcccaggctgaggacgaagccgattacta ttgcagctcccgggataagagcggctccagactgagcgtgttcggaggaggaactaaactgaccgtcctcagtcagccca aggctgccccctcggtcactctgttcccgccctcgagtgaggagcttcaagccaacaaggccacactggtgtgtctcata agtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccaccac accctccaaacaaagcaacaacaagtacgcggccagcagctacctgagcctgacgcctgancagtggaangtcccacaga agctacagctgccaggtcacgcatgaagggagcaccgt

SEQ ID NO: 26 is the amino acid sequence of HIV-1 Env from the HXB2 strain of HIV-1.

SEQ ID NOs: 27-33 are amino acid sequences of the Vh and Vl domains of the VRC01, VRC07,

VRC07-523, and VRC07-544 antibodies.

SEQ ID NO: 34 is the amino acid sequence of a MPER peptide.

SEQ ID NO: 35 is the amino acid sequence of the H6-51 l-4mut-S100cW Vh.

LYLEMNNVRTEDTGYYFCARTGKYYDFWWGYPPGEEYFQDWGQGTLVIVSS

SEQ ID NO: 36 is the amino acid sequence of the H6-51 l-4mut-DS Vh.

LYLEMNNVRTEDTGYYFCARTGKYYDFWSGCPPGEEYFQDWGQGTLVIVSS

SEQ ID NO: 37 is the amino acid sequence of the H6-511-4mut-S100cW-DS Vh.

LYLEMNNVRTEDTGYYFCARTGKYYDFWWGCPPGEEYFQDWGQGTLVIVSS

SEQ ID NO: 38 is the amino acid sequence of the HC6-S74Y-511-S100cW V_H.

LYLEMNNLRTEDSGLYFCARTGKYYDFWWGYPPGEEYFQDWGRGTLVIVSS

SEQ ID NO: 39 is the amino acid sequence of the HC6-S74Y-511-DS Vh.

LYLEMNNLRTEDSGLYFCARTGKYYDFWSGCPPGEEYFQDWGRGTLVIVSS

SEQ ID NO: 40 is the amino acid sequence of the HC6-S74Y-511-S100cW-DS V_H.

LYLEMNNLRTEDSGLYFCARTGKYYDFWWGCPPGEEYFQDWGRGTLVIVSS

SEQ ID NO: 41 is the amino acid sequence of the rL3-6mut-DS Vl.

ASELTQDPAVSVALKQTVTITCRGDSLRCHYASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSGSASGNRASLTITGAQAE

DEADYYCSSRDKSGSRLSVFGGGTKLTVL

SEQ ID NO: 42 is the amino acid sequence of the rL3-6mut-Y32W Vl.

ASELTQDPAVSVALKQTVTITCRGDSLRSHWASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSGSASGNRASLTITGAQAE

DEADYYCSSRDKSGSRLSVFGGGTKLTVL

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SEQ ID NO: 43 is the amino acid sequence of the rL3-6mut-Y32F VlASELTQDPAVSVALKQTVTITCRGDSLRSHFASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSGSASGNRASLTITGAQAE

DEADYYCSSRDKSGSRLSVFGGGTKLTVL

SEQ ID NO: 44 is an exemplary nucleic acid sequence encoding the HC6-S74Y-511 Vhgaagtgcggctggctgagagcggcggggggctggtcaaacctggcgggtcactgcggctgtcctgttctgcctccggctt cgattttgataacgcatggatgacatgggtgcgacagccacctggaaaggggctggagtgggtcggcagaatcactggac ctggcgaagggtggtctgtggactacgcagctccagtcgagggacgattcaccattagtagagataactacaagaataca ctgtatctggagatgaacaatctgaggactgaagacagcggcctgtatttctgcgcccgcaccgggaaatactatgattt ttggtctgggtacccacccggagaggaatattttcaggactggggacggggcaccctggtgatcgtcagctcc

SEQ ID NO: 45 is an exemplary nucleic acid sequence encoding the H6-51 l-4mut Vh.

gaggttagactggtggagtcaggaggggggcttgtgaagcccggtgggtctctccgcctgagctgttctgcctccggctt tgatttcgataacgcctggatgacctgggtcaggcagcctccaggtaagggactggagtgggtgggaagaatcacaggtc caggcgagggctggtccgtggactacgcggaatctgttaaagggcggtttacaatctcaagggacaataccaagaatacc ttgtatttggagatgaacaacgtgagaactgaagacaccggatattacttctgtgccagaacaggcaaatactacgactt ctggtccggctatccccctggcgaggaatattttcaagactggggtcagggaacccttgttatcgtgtcctcc

SEQ ID NO: 46 is an exemplary nucleic acid sequence encoding the rL3 Vl.

tcatacgaactgactcaggacactggcgtctctgtggcactggggaggactgtgactattacttgccgaggcgactcact gcggagccactacgcttcctggtatcagaagaaacccggccaggcacctgtgctgctgttctacggaaagaacaataggc catctggcatccccgaccgcttttctggcagtgcatcagggaaccgagccagtctgaccattaccggcgcccaggctgag gacgaagccgattactattgcagctcccgggataagagcggctccagactgagcgtgttcggaggaggaactaaactgac cgtcctc

SEQ ID NO: 47 is an exemplary nucleic acid sequence encoding the rL3-6mut Vl.

accaccatgggatggtcatgtatcatcctttttctagtagcaactgcaaccggttctgtgaccgcatccgaactgactca ggaccctgccgtctctgtggcactgaagcagactgtgactattacttgccgaggcgactcactgcggagccactacgctt cctggtatcagaagaaacccggccaggcacctgtgctgctgttctacggaaagaacaataggccatctggcatccccgac cgcttttctggcagtgcatcagggaaccgagccagtctgaccattaccggcgcccaggctgaggacgaagccgattacta ttgcagctcccgggataagagcggctccagactgagcgtgttcggaggaggaactaaactgaccgtcctc

SEQ ID NO: 48 is an exemplary nucleic acid sequence of an expression vector encoding the HC6S74Y-511 V_H.

SEQ ID NO: 49 is an exemplary nucleic acid sequence of an expression vector encoding the H65H-4mut Vh.

SEQ ID NO: 50 is an exemplary nucleic acid sequence of an expression vector encoding the rL3 V_L.

SEQ ID NO: 51 is an exemplary nucleic acid sequence of an expression vector encoding the rL36mut VlSEQ ID NOs: 52-57 are the amino acid sequences of IMGT CDRs.

SEQ ID NO: 58 is the amino acid sequence of the rL3-6mut-DS-Y32W Vl.

ASELTQDPAVSVALKQTVTITCRGDSLRCHWASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSGSASGNRASLTITGAQAE

DEADYYCSSRDKSGSRLSVFGGGTKLTVL

SEQ ID NO: 59 is the amino acid sequence of the rL3-6mut-DS-Y32F Vl.

ASELTQDPAVSVALKQTVTITCRGDSLRCHFASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSGSASGNRASLTITGAQAE

DEADYYCSSRDKSGSRLSVFGGGTKLTVL

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SEQ ID NOs: 60-63 are an exemplary nucleic acid sequence of expression vectors encoding the

10E8v4-DS V_H, the 10E8v4-DS (or 10E8v4-100cW-DS) VL, the 10E8v4-100cW V_H, or the 10E8v4lOOcW-DS Vh, respectively.

SEQ ID NO: 64 is an exemplary nucleic acid sequence encoding the H6-51 l-4mut-S100cW Vh (Seq_35).

atgggctggtcctgtattatcctgttcctggtcgcaactgctactggcgtccattcagaagtgaggctggtcgagagcgg cggcggcctggtgaagccaggaggaagcctgcgactgagctgctccgcctctggcttcgactttgataacgcttggatga catgggtgcgacagccccctggaaaaggcctggagtgggtcggaagaatcaccggccccggagagggatggagtgtggac tacgcagaatcagtcaagggccggttcaccattagccgggataacaccaaaaatacactgtatctggagatgaacaatgt caggactgaagacaccgggtactatttctgtgcccgcaccggaaagtactatgatttttggtggggctacccacccggag aagaatactttcaggactggggacagggaacactggtcatcgtcagcagc

SEQ ID NO: 65 is an exemplary nucleic acid sequence encoding the H6-51 l-4mut-DS Vh (Seq_36).

atgggatggtcatgtatcatcctttttctagtagcaactgcaaccggtgtacattctgaggttagactggtggagtcagg aggggggcttgtgaagcccggtgggtctctccgcctgagctgttctgcctccggctttgatttcgataacgcctggatga cctgggtcaggcagcctccaggtaagggactggagtgggtgggaagaatcacaggtccaggcgagggctggtccgtggac tacgcggaatctgttaaagggcggtttacaatctcaagggacaataccaagaataccttgtatttggagatgaacaacgt gagaactgaagacaccggatattacttctgtgccagaacaggcaaatactacgacttctggtccggctgcccccctggcg aggaatattttcaagactggggtcagggaacccttgttatcgtgtcctcc

SEQ ID NO: 66 is an exemplary nucleic acid sequence encoding the H6-511-4mut-S100cW-DS Vh (Seq_37).

atgggatggtcatgtatcatcctttttctagtagcaactgcaaccggtgtacattctgaggttagactggtggagtcagg aggggggcttgtgaagcccggtgggtctctccgcctgagctgttctgcctccggctttgatttcgataacgcctggatga cctgggtcaggcagcctccaggtaagggactggagtgggtgggaagaatcacaggtccaggcgagggctggtccgtggac tacgcggaatctgttaaagggcggtttacaatctcaagggacaataccaagaataccttgtatttggagatgaacaacgt gagaactgaagacaccggatattacttctgtgccagaacaggcaaatactacgacttctggtggggctgcccccctggcg aggaatattttcaagactggggtcagggaacccttgttatcgtgtcctcc

SEQ ID NO: 67 is an exemplary nucleic acid sequence encoding the HC6-S74Y-511-S100cW Vh (Seq_38).

gaagtgcggctggctgagagcggcggggggctggtcaaacctggcgggtcactgcggctgtcctgttctgcctccggctt cgattttgataacgcatggatgacatgggtgcgacagccacctggaaaggggctggagtgggtcggcagaatcactggac ctggcgaagggtggtctgtggactacgcagctccagtcgagggacgattcaccattagtagagataactacaagaataca ctgtatctggagatgaacaatctgaggactgaagacagcggcctgtatttctgcgcccgcaccgggaaatactatgattt ttggtgggggtacccacccggagaggaatattttcaggactggggacggggcaccctggtgatcgtcagctcc

SEQ ID NO: 68 is an exemplary nucleic acid sequence encoding the HC6-S74Y-511-DS Vh (Seq_39).

Gaagtgcggctggctgagagcggcggggggctggtcaaacctggcgggtcactgcggctgtcctgttctgcctccggctt cgattttgataacgcatggatgacatgggtgcgacagccacctggaaaggggctggagtgggtcggcagaatcactggac ctggcgaagggtggtctgtggactacgcagctccagtcgagggacgattcaccattagtagagataactacaagaataca ctgtatctggagatgaacaatctgaggactgaagacagcggcctgtatttctgcgcccgcaccgggaaatactatgattt ttggtctgggtgcccacccggagaggaatattttcaggactggggacggggcaccctggtgatcgtcagctcc

SEQ ID NO: 69 is an exemplary nucleic acid sequence of the HC6-S74Y-511-S100cW-DS Vh (Seq_40).

gaagtgcggctggctgagagcggcggggggctggtcaaacctggcgggtcactgcggctgtcctgttctgcctccggctt cgattttgataacgcatggatgacatgggtgcgacagccacctggaaaggggctggagtgggtcggcagaatcactggac

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PCT/US2016/060390 ctggcgaagggtggtctgtggactacgcagctccagtcgagggacgattcaccattagtagagataactacaagaataca ctgtatctggagatgaacaatctgaggactgaagacagcggcctgtatttctgcgcccgcaccgggaaatactatgattt ttggtgggggtgcccacccggagaggaatattttcaggactggggacggggcaccctggtgatcgtcagctcc

SEQ ID NO: 70 is an exemplary nucleic acid sequence encoding the rL3-6mut-DS Vl (Seq_41).

atgggatggtcatgtatcatcctttttctagtagcaactgcaaccggttctgtgaccgcatccgaactgactcaggaccc tgccgtctctgtggcactgaagcagactgtgactattacttgccgaggcgactcactgcggtgccactacgcttcctggt atcagaagaaacccggccaggcacctgtgctgctgttctacggaaagaacaataggccatctggcatccccgaccgcttt tctggcagtgcatcagggaaccgagccagtctgaccattaccggcgcccaggctgaggacgaagccgattactattgcag ctcccgggataagagcggctccagactgagcgtgttcggaggaggaactaaactgaccgtcctc

SEQ ID NO: 71 is an exemplary nucleic acid sequence encoding the rL3-6mut-Y32W Vl (Seq_42).

atgggatggtcatgtatcatcctttttctagtagcaactgcaaccggttctgtgaccgcatccgaactgactcaggaccc tgccgtctctgtggcactgaagcagactgtgactattacttgccgaggcgactcactgcggagccactgggcttcctggt atcagaagaaacccggccaggcacctgtgctgctgttctacggaaagaacaataggccatctggcatccccgaccgcttt tctggcagtgcatcagggaaccgagccagtctgaccattaccggcgcccaggctgaggacgaagccgattactattgcag ctcccgggataagagcggctccagactgagcgtgttcggaggaggaactaaactgaccgtcctc

SEQ ID NO: 72 is the amino acid sequence encoding rL3-6mut-Y32F Vl (Seq_43).

atgggatggtcatgtatcatcctttttctagtagcaactgcaaccggttctgtgaccgcatccgaactgactcaggaccc tgccgtctctgtggcactgaagcagactgtgactattacttgccgaggcgactcactgcggagccactttgcttcctggt atcagaagaaacccggccaggcacctgtgctgctgttctacggaaagaacaataggccatctggcatccccgaccgcttt tctggcagtgcatcagggaaccgagccagtctgaccattaccggcgcccaggctgaggacgaagccgattactattgcag ctcccgggataagagcggctccagactgagcgtgttcggaggaggaactaaactgaccgtcctc

SEQ ID NO: 73 is an exemplary nucleic acid sequence encoding rL3-6mut-DS-Y32W Vl (Seq_58).

SEQ ID NO: 74 is an exemplary nucleic acid sequence encoding rL3-6mut-DS-Y32F Vl (Seq_59).

SEQ ID NO: 75 is the amino acid sequence of the H6-51 l-4mut-S100cF Vh.

LYLEMNNVRTEDTGYYFCARTGKYYDFWFGYPPGEEYFQDWGQGTLVIVSS

SEQ ID NO: 76 is the amino acid sequence of the H6-51 l-4mut V5R SlOOcF Vh.

EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAESVKGRFTISRDNTKNT

LYLEMNNVRTEDTGYYFCARTGKYYDFWFGYPPGEEYFQDWGQGTLVIVSS

SEQ ID NO: 77 is the amino acid sequence of the HCDR3 of H6-511-4mut-S100cF V_H(IMGT).

SEQ ID NO: 78 is an exemplary nucleic acid sequence encoding H6-511-4mut-S100cF Vh (Seq_75).

gaggttagactggtggagtcaggaggggggcttgtgaagcccggtgggtctctccgcctgagctgttctgcctccggctt tgatttcgataacgcctggatgacctgggtcaggcagcctccaggtaagggactggagtgggtgggaagaatcacaggtc caggcgagggctggtccgtggactacgcggaatctgttaaagggcggtttacaatctcaagggacaataccaagaatacc

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PCT/US2016/060390 ttgtatttggagatgaacaacgtgagaactgaagacaccggatattacttctgtgccagaacaggcaaatactacgactt ctggttcggctatccccctggcgaggaatattttcaagactggggtcagggaacccttgttatcgtgtcctcc

SEQ ID NO: 79 is an exemplary nucleic acid sequence encoding H6-511-4mut V5R SlOOcF Vh (Seq_76).

gaggttagactgcgggagtcaggaggggggcttgtgaagcccggtgggtctctccgcctgagctgttctgcctccggctt tgatttcgataacgcctggatgacctgggtcaggcagcctccaggtaagggactggagtgggtgggaagaatcacaggtc caggcgagggctggtccgtggactacgcggaatctgttaaagggcggtttacaatctcaagggacaataccaagaatacc ttgtatttggagatgaacaacgtgagaactgaagacaccggatattacttctgtgccagaacaggcaaatactacgactt ctggttcggctatccccctggcgaggaatattttcaagactggggtcagggaacccttgttatcgtgtcctcc

SEQ ID NOs: 80-81 are nucleic acid sequence of expression vectors encoding the H6-511-4mut- SlOOcF and H6-511-4mut V5R SlOOcF Vh regions, respectively.

SEQ ID NO: 82 is the amino acid sequence of the H6-511-4mut-S100cF Vh linked to an IgGl constant region.

EVRLVESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAESVKGRFTISRDNTKNT LYLEMNNVRTEDTGYYFCARTGKYYDFWFGYPPGEEYFQDWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 83 is the amino acid sequence of the H6-511-4mut-V5R SlOOcF Vnlinked to an IgGl constant region.

EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAESVKGRFTISRDNTKNT LYLEMNNVRTEDTGYYFCARTGKYYDFWFGYPPGEEYFQDWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 84 is the amino acid sequence of the rF3-6mut Vl linked to an Ig lambda constant region.

ASELTQDPAVSVALKQTVTITCRGDSLRSHYASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSGSASGNRASLTITGAQAE

DEADYYCSSRDKSGSRLSVFGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA

GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

DETAILED DESCRIPTION

I. Summary of Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishers, 2009; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley-VCH in 16 volumes, 2008; and other similar references.

As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes single or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term

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PCT/US2016/060390 “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various embodiments, the following explanations of terms are provided:

10E8: A neutralizing antibody that specifically binds to an epitope on the MPER of gp41. The 10E8 antibody and its binding to gp41 have been previously described, see, e.g., Huang et al. Nature, 491: 406-412, 2012, which is incorporated by reference herein in its entirety. The amino acid sequences of the heavy and light variable regions of the 10E8 antibody are set forth as SEQ ID NOs: 1 and 2.

Administration: The introduction of a composition into a subject by a chosen route. Administration can be local or systemic. For example, if the chosen route is intravenous, the composition is administered by introducing the composition into a vein of the subject. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (for example, topical), intranasal, vaginal, and inhalation routes.

Amino acid substitution: The replacement of one amino acid in peptide with a different amino acid.

Anti-retroviral agent: An agent that specifically inhibits a retrovirus from replicating or infecting cells. Non-limiting examples of antiretroviral drugs include entry inhibitors (e.g., enfuvirtide), CCR5 receptor antagonists (e.g., aplaviroc, vicriviroc, maraviroc), reverse transcriptase inhibitors (e.g., lamivudine, zidovudine, abacavir, tenofovir, emtricitabine, efavirenz), protease inhibitors (e.g., lopivar, ritonavir, raltegravir, darunavir, atazanavir), maturation inhibitors (e.g., alpha interferon, bevirimat and vivecon).

Anti-retroviral therapy (ART): A therapeutic treatment for HIV-1 infection involving administration of at least one anti-retroviral agents (e.g., one, two, three or four anti-retroviral agents) to an HIV-1 infected individual. One example of an ART regimen includes treatment with a combination of tenofovir, emtricitabine and efavirenz. In some examples, ART includes Highly Active Anti-Retroviral Therapy (HAART). One example of a HAART regimen includes treatment with a combination of tenofovir, emtricitabine and efavirenz.

Antibody: An immunoglobulin, antigen-binding fragment, or derivative thereof, that specifically binds and recognizes an analyte (antigen) such as HIV-1 gp41. The term “antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

Non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof known in the art that retain binding affinity for the antigen. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; singlechain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.

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Antibody fragments include antigen binding fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and

Dubel (Ed), Antibody Engineering, Vols. 1-2, 2^nd Ed., Springer Press, 2010).

A single-chain antibody (scFv) is a genetically engineered molecule containing the Vh and Vl domains of one or more antibody(ies) linked by a suitable polypeptide linker as a genetically fused single chain molecule (see, for example, Bird et al., Science, 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci., 85:5879-5883, 1988; Ahmad etal., Clin. Dev. Immunol., 2012, doi: 10.1155/2012/980250; Marbry, IDrugs, 13:543-549, 2010). The intramolecular orientation of the VH-domain and the VL-domain in a scFv, is typically not decisive for scFvs. Thus, scFvs with both possible arrangements (VH-domain-linker domainVL-domain; VL-domain-linker domain-VH-domain) may be used.

In a dsFv the Vh and Vl have been mutated to introduce a disulfide bond to stabilize the association of the chains. Diabodies also are included, which are bivalent, bispecific antibodies in which Vh and Vl domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see, for example, Holliger et al., Proc.

Natl. Acad. Sci., 90:6444-6448, 1993; Poljak et al., Structure, 2:1121-1123, 1994).

Antibodies also include genetically engineered forms such as chimeric antibodies (such as humanized murine antibodies) and heteroconjugate antibodies (such as bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3^rdEd., W.H. Freeman & Co., New York, 1997.

An “antibody that binds to the same epitope” as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more. Antibody competition assays are known, and an exemplary competition assay is provided herein.

An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For instance, a naturallyoccurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, while a bispecific or bifunctional antibody has two different binding sites.

Typically, a naturally occurring immunoglobulin has heavy chains and light chains interconnected by disulfide bonds. Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable domain genes. There are two types of light chain, lambda (λ) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variable region (see, e.g., Kindt et al.

Kuby Immunology, 6^th ed., W.H. Freeman and Co., page 91 (2007).) References to “Vh” or “VH” refer to the variable region of an antibody heavy chain, including that of an antigen binding fragment, such as Fv,

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PCT/US2016/060390 scFv, dsFv or Fab. References to “Vl” or “VL” refer to the variable domain of an antibody light chain, including that of an Fv, scFv, dsFv or Fab. In several embodiments, the Vh and Vl combine to specifically bind the antigen. In additional embodiments, only the Vh is required. For example, naturally occurring camelid antibodies consisting of a heavy chain only are functional and stable in the absence of light chain (see, e.g., Hamers-Casterman etal., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996). Any of the disclosed antibodies comprises a heterologous constant regon. For example the antibody comprises a constant region that is different from a native constant region, such as a constant region including one or more modifications (such as the “LS” mutations) to increase half-life.

The Vh and Vl contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs” (see, e.g., Rabat etal., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.

The CDRs are primarily responsible for binding to an epitope of an antigen. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including those described by Rabat et al. (“Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991; “Rabat” numbering scheme), Al-Lazikani et al., (JMB 273,927-948, 1997; “Chothia” numbering scheme), and Lefranc et al. (“IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev. Comp. Immunol., 27:55-77, 2003; “IMGT” numbering scheme). The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (from the N-terminus to C-terminus), and are also typically identified by the chain in which the particular CDR is located. Thus, a Vh CDR3 is the CDR3 from the Vh of the antibody in which it is found, whereas a Vl CDR1 is the CDR1 from the Vl of the antibody in which it is found. Light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are sometimes referred to as HCDR1, HCDR2, and HCDR3.

A “monoclonal antibody” is an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, for example, containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic

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PCT/US2016/060390 animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein. In some examples monoclonal antibodies are isolated from a subject. Monoclonal antibodies can have conservative amino acid substitutions, which have substantially no effect on antigen binding or other immunoglobulin functions.

(See, for example, Harlow & Lane, Antibodies, A Laboratory Manual, 2^nd ed. Cold Spring Harbor Publications, New York (2013).) In some embodiments, any of the antibodies disclosed herein can be a monoclonal antibody.

A “humanized” antibody or antigen binding fragment includes a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) antibody or antigen binding fragment. The non-human antibody or antigen binding fragment providing the CDRs is termed a “donor,” and the human antibody or antigen binding fragment providing the framework is termed an “acceptor.” In one embodiment, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they can be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized antibody or antigen binding fragment, except possibly the CDRs, are substantially identical to corresponding parts of natural human antibody sequences.

A “chimeric antibody” is an antibody which includes sequences derived from two different antibodies, and are typically of different species. In some examples, a chimeric antibody includes one or more CDRs and/or framework regions from one human antibody and CDRs and/or framework regions from another human antibody.

A “fully human antibody” or “human antibody” is an antibody, which includes sequences from (or derived from) the human genome, and does not include sequence from another species. In some embodiments, a human antibody includes CDRs, framework regions, and (if present) an Fc region from (or derived from) the human genome. Human antibodies can be identified and isolated using technologies for creating antibodies based on sequences derived from the human genome, for example by phage display or using transgenic animals (see, e.g., Barbas etal. Phage display: A Laboratory Manuel. 1^st Ed. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23: 1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20:450-459, 2008)

Antibody or antigen binding fragment that neutralizes HIV-1: An antibody or antigen binding fragment that specifically binds to HIV-1 Env (e.g., that binds to gp41) in such a way as to inhibit a biological function associated with HIV-1 Env (such as binding to its target receptor). In several embodiments, an antibody or antigen binding fragment that neutralizes HIV-1 reduces the infectious titer of HIV-1. In some embodiments, an antibody or antigen binding fragment that specifically binds to HIV-1 Env can neutralize two or more (such as 3, 4, 5, 6, 7, 8, 9, 10, or more) strains of HIV-1.

Broadly neutralizing antibodies to HIV-1 are distinct from other antibodies to HIV-1 in that they neutralize a high percentage of the many types of HIV-1 in circulation. Thus, a HIV-1 broadly neutralizing antibody is an antibody that reduces the infectious titer of HIV-1 by binding to and inhibiting the function of

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PCT/US2016/060390 related HIV-1 antigens, such as antigens that share at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with antigenic surface of the antigen. In some embodiments, broadly neutralizing antibodies to HIV-1 are distinct from other antibodies to HIV-1 in that they neutralize a high percentage (such as at least 80%, at least 85%, at least 90%, or at least 95%) of the many types of HIV-1 in circulation.

Antibody self-reactivity or autoreactivity: A property of an antibody, whereby the antibody reacts with self-epitopes, that is epitopes of proteins and/or lipids that are produced by the subject. An antibody that does not have self-reactivity does not substantially bind to epitopes or lipids present on the membrane of a cell from a subject. Methods of determining if an antibody reacts with self epitopes are known to the person of ordinary skill in the art. In one example, antibody self reactivity is evaluated using HEp-2 cell staining, a cardiolipin binding assay, or an anti-nuclear antigen (ANA) assay. The anti-ANA assay comprises an anti-ANA LUMINEX® assay or an ANA cell-staining assay, for example. In several embodiments, a disclosed antibody is not self-reactive (or autoreactive), or is minimally self-reactive. In one non-limiting example, a disclosed antibody is not substantially more self-reactive that the 10E8 antibody.

For example the disclosed antibody or antigen binding fragment can have no more than 10% greater autoreactivity compared to the 10E8 antibody, for example as measured using HEp-2 cell staining, cardiolipin binding, an anti-ANA LUMINEX® assay, or an ANA cell-staining assay. In another nonlimiting example, a disclosed antibody noes not have self reactivity above background levels, for example, as measured using an anti-ANA LUMINEX® assay or an ANA cell-staining assay.

Biological sample: A sample obtained from a subject. Biological samples include all clinical samples useful for detection of disease or infection (for example, HIV-1 infection) in subjects, including, but not limited to, cells, tissues, and bodily fluids, such as blood, derivatives and fractions of blood (such as serum), cerebrospinal fluid; as well as biopsied or surgically removed tissue, for example tissues that are unfixed, frozen, or fixed in formalin or paraffin. In a particular example, a biological sample is obtained from a subject having or suspected of having an HIV-1 infection.

Bispecific antibody: A recombinant molecule composed of two different antigen binding domains that consequently binds to two different antigenic epitopes. Bispecific antibodies include chemically or genetically linked molecules of two antigen-binding domains. The antigen binding domains can be linked using a linker. The antigen binding domains can be monoclonal antibodies, antigen-binding fragments (e.g., Fab, scFv), or combinations thereof. A bispecific antibody comprises one or more constant domains, but does not necessarily include a constant region.

Conditions sufficient to form an immune complex: Conditions which allow an antibody or antigen binding fragment to bind to its cognate epitope to a detectably greater degree than, and/or to the substantial exclusion of, binding to substantially all other epitopes. Conditions sufficient to form an immune complex are dependent upon the format of the binding reaction and typically are those utilized in immunoassay protocols or those conditions encountered in vivo. See Harlow & Lane, Antibodies, A Laboratory Manual, 2^nd ed. Cold Spring Harbor Publications, New York (2013), for a description of immunoassay formats and conditions. The conditions employed in the methods are “physiological

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PCT/US2016/060390 conditions” which include reference to conditions (e.g., temperature, osmolarity, pH) that are typical inside a living mammal or a mammalian cell. While it is recognized that some organs are subject to extreme conditions, the intra-organismal and intracellular environment normally lies around pH 7 (e.g., from pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), contains water as the predominant solvent, and exists at a temperature above 0°C and below 50°C. Osmolarity is within the range that is supportive of cell viability and proliferation.

The formation of an immune complex can be detected through conventional methods known to the skilled artisan, for instance immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (for example, Western blot), magnetic resonance imaging, CT scans, X-ray and affinity chromatography. Immunological binding properties of selected antibodies may be quantified using methods well known in the art.

Conjugate: A complex of two molecules linked together, for example, linked together by a covalent bond. In one embodiment, an antibody is linked to an effector molecule; for example, an antibody that specifically binds to HIV-1 Env covalently linked to an effector molecule. The linkage can be by chemical or recombinant means. In one embodiment, the linkage is chemical, wherein a reaction between the antibody moiety and the effector molecule has produced a covalent bond formed between the two molecules to form one molecule. A peptide linker (short peptide sequence) can optionally be included between the antibody and the effector molecule. Because conjugates can be prepared from two molecules with separate functionalities, such as an antibody and an effector molecule, they are also sometimes referred to as “chimeric molecules.”

Conservative variants: “Conservative” amino acid substitutions are those substitutions that do not substantially affect or decrease a function of a protein, such as the ability of the protein to interact with a target protein. For example, an HIV-specific antibody comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 conservative substitutions compared to a reference antibody sequence and retain specific binding activity for HIV-1 antigen, and/or HIV-1 neutralization activity. The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid.

Furthermore, one of ordinary skill will recognize that individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (for instance less than 5%, in some embodiments less than 1%) in an encoded sequence are conservative variations where the alterations result in the substitution of an amino acid with a chemically similar amino acid.

Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

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5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Non-conservative substitutions are those that reduce an activity or function of the HIV-specific antibody, such as the ability to specifically bind to gp41. For instance, if an amino acid residue is essential for a function of the protein, even an otherwise conservative substitution may disrupt that activity. Thus, a conservative substitution does not alter the basic function of a protein of interest.

Contacting: Placement in direct physical association; includes both in solid and liquid form, which can take place either in vivo or in vitro. Contacting includes contact between one molecule and another molecule, for example the amino acid on the surface of one polypeptide, such as an antigen, that contacts another polypeptide, such as an antibody. Contacting can also include contacting a cell for example by placing an antibody in direct physical association with a cell.

Control: A reference standard. In some embodiments, the control is a negative control, such as sample obtained from a healthy patient not infected with HIV-1. In other embodiments, the control is a positive control, such as a tissue sample obtained from a patient diagnosed with HIV-1 infection. In still other embodiments, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of HIV-1 patients with known prognosis or outcome, or group of samples that represent baseline or normal values).

A difference between a test sample and a control can be an increase or conversely a decrease. The difference can be a qualitative difference or a quantitative difference, for example a statistically significant difference. In some examples, a difference is an increase or decrease, relative to a control, of at least about 5%, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, or at least about 500%.

Degenerate variant: In the context of the present disclosure, a “degenerate variant” refers to a polynucleotide encoding a protein (for example, an antibody that specifically binds gp41 or variable region thereof) that includes a sequence that is degenerate as a result of the genetic code. There are twenty natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included as long as the amino acid sequence of the antibody that binds gp41 encoded by the nucleotide sequence is unchanged.

Detectable marker: A detectable molecule (also known as a label) that is conjugated directly or indirectly to a second molecule, such as an antibody, to facilitate detection of the second molecule. For example, the detectable marker can be capable of detection by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (such as CT scans, MRIs, ultrasound, fiberoptic examination, and laparoscopic examination). Specific, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI). In one example, a “labeled

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PCT/US2016/060390 antibody” refers to incorporation of another molecule in the antibody. For example, the label is a detectable marker, such as the incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (such as ³⁵S or ¹³¹I), fluorescent labels (such as fluorescein isothiocyanate (F1TC), rhodamine, lanthanide phosphors), enzymatic labels (such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (such as a leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), or magnetic agents, such as gadolinium chelates. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance. Methods for using detectable markers and guidance in the choice of detectable markers appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4^th ed, Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013).

Detecting: To identify the existence, presence, or fact of something. General methods of detecting are known to the skilled artisan and may be supplemented with the protocols and reagents disclosed herein. For example, included herein are methods of detecting a cell that expresses gp41 in a subject.

Effector molecule: A molecule intended to have or produce a desired effect; for example, a desired effect on a cell to which the effector molecule is targeted. Effector molecules comprises, for example, polypeptides and small molecules. In one non-limiting example, the effector molecule is a toxin. IJie skilled artisan will understand that some effector molecules may have or produce more than one desired effect.

Epitope: An antigenic determinant. IJiese are particular chemical groups or peptide sequences on a molecule that are antigenic, i.e. that elicit a specific immune response. An antibody specifically binds a particular antigenic epitope on a polypeptide. In some examples a disclosed antibody specifically binds to an epitope on gp41.

Expression: Transcription or translation of a nucleic acid sequence. For example, an encoding nucleic acid sequence (such as a gene) can be expressed when its DNA is transcribed into an RNA or RNA fragment, which in some examples is processed to become mRNA. An encoding nucleic acid sequence (such as a gene) may also be expressed when its mRNA is translated into an amino acid sequence, such as a protein or a protein fragment. In a particular example, a heterologous gene is expressed when it is transcribed into an RNA. In another example, a heterologous gene is expressed when its RNA is translated into an amino acid sequence. Regulation of expression comprises controls on transcription, translation,

RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization or degradation of specific protein molecules after they are produced.

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Expression Control Sequences: Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus expression control sequences comprises appropriate promoters, enhancers, transcription terminators, a start codon (ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term “control sequences” is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences comprises a promoter.

A promoter is a minimal sequence sufficient to direct transcription. Also included are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for celltype specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters are included (see for example, Bitter etal., Methods inEnzymology 153:516-544, 1987). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used. In one embodiment, when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (such as metallothionein promoter) or from mammalian viruses (such as the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) can be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences.

A polynucleotide can be inserted into an expression vector that contains a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host. The expression vector typically contains an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of the transformed cells.

Expression vector: A vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

Fc region: The polypeptide including the constant region of an antibody excluding the first constant immunoglobulin domain. Fc region generally refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM. An Fc region may also include part or all of the flexible hinge N-terminal to these domains. For IgA and IgM, an Fc region may or may not include the tailpiece, and may or may not be bound by the J chain. For IgG, the Fc region includes immunoglobulin domains Cy2 and Cy3 and the lower part of the hinge between Cyl and

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Cy2. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat. For IgA, the Fc region includes immunoglobulin domains Ca2 and Ca3 and the lower part of the hinge between Cal and Ca2. Any of the disclosed antibodies comprises a heterologous Fc region or heterologous constant domain. For example the antibody comprises a Fc region or constant domain that is different from a native Fc region or constant domain, such as a Fc region or constant domain including one or more modifications (such as the “LS” mutations) to increase half-life.

HIV-1 Envelope protein (Env): The HIV-1 envelope protein is initially synthesized as a precursor protein of 845-870 amino acids in size, designated gpl60. Individual gpl60 polypeptides form a homotrimer and undergo glycosylation within the Golgi apparatus as well as processing to remove the signal peptide, and cleavage by a cellular protease between approximately positions 511/512 to generate separate gpl20 and gp41 polypeptide chains, which remain associated as gpl20/gp41 protomers within the homotrimer. The ectodomain (that is, the extracellular portion) of the HIV-1 Env trimer undergoes several structural rearrangements from a prefusion mature (cleaved) closed conformation that evades antibody recognition, through intermediate conformations that bind to receptors CD4 and co-receptor (either CCR5 or CXCR4), to a postfusion conformation.

The numbering used in the disclosed HIV-1 Env proteins and fragments thereof is relative to the HXB2 numbering scheme as set forth in Numbering Positions in HIV Relative to HXB2CG Bette Korber et al., Human Retroviruses and AIDS 1998: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Korber etal., Eds. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM, which is incorporated by reference herein in its entirety.

HIV-1 gpl20: A polypeptide that is part of the HIV-1 Env protein. Mature gpl20 includes approximately HIV-1 Env residues 31-511, contains most of the external, surface-exposed, domains of the HIV-1 Env trimer, and it is gpl20 which binds both to cellular CD4 receptors and to cellular chemokine receptors (such as CCR5). A mature gpl20 polypeptide is a heavily N-glycosylated extracellular polypeptide that interacts with the gp41 ectodomain to form an HIV-1 Env protomer that trimerizes to form the HIV-1 Env trimer.

HIV-1 gp41: A polypeptide that is part of the HIV-1 Env protein. Mature gp41 includes approximately HIV-1 Env residues 512-860, and includes cytosolic-, transmembrane-, and ecto-domains. The gp41 ectodomain (including approximately HIV-1 Env residues 512-644) can interact with gpl20 to form an HIV-1 Env protomer that trimerizes to form the HIV-1 Env trimer.

Gp41 includes the membrane-proximal external region (MPER), which is immediately Nterminal of the transmembrane region of gp41. The MPER is highly hydrophobic (50% of residues are hydrophobic) and is highly conserved across many HIV-1 clades (Zwick, M.B., et al., J Virol, 75 (22): p. 10892-905, 2001). The MPER of HIV-1 gp41 includes the target epitope of the 10E8 monoclonal antibody.

Human Immunodeficiency Virus type 1 (HIV-1): A retrovirus that causes immunosuppression in humans (HIV-1 disease), and leads to a disease complex known as the acquired immunodeficiency

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PCT/US2016/060390 syndrome (AIDS). “HIV-1 disease” refers to a well-recognized constellation of signs and symptoms (including the development of opportunistic infections) in persons who are infected by an HIV-1 virus, as determined by antibody or western blot studies. Laboratory findings associated with this disease include a progressive decline in T cells. Related viruses that are used as animal models include simian immunodeficiency virus (SIV) and feline immunodeficiency virus (FIV). Treatment of HIV-1 with HAART has been effective in reducing the viral burden and ameliorating the effects of HIV-1 infection in infected individuals.

HXB2 numbering system: A reference numbering system for HIV-1 protein and nucleic acid sequences, using HIV-1 HXB2 strain sequences as a reference for all other HIV-1 strain sequences. The person of ordinary skill in the art is familiar with the HXB2 numbering system, and this system is set forth in “Numbering Positions in HIV Relative to HXB2CG,” Bette Korber etal., Human Retroviruses and AIDS 1998: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Korber B, Kuiken CL,

Foley B, Hahn B, McCutchan F, Mellors JW, and Sodroski J, Eds. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM, which is incorporated by reference herein in its entirety. HXB2 is also known as: HXBc2, for HXB clone 2; HXB2R, in the Los Alamos HIV database, with the R for revised, as it was slightly revised relative to the original HXB2 sequence; and HXB2CG in GENBANK™, for HXB2 complete genome. The numbering used in gpl20 and gp41 polypeptides disclosed herein is relative to the HXB2 numbering scheme. For reference, the amino acid sequence of HIV-1 Env of HXB2 is set forth below:

MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATH ACVPTDPNPQEWLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKLTPLCVSLKCTDLKNDTNTNSSS GRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYKLTSCNTSVITQACPKVSFEPIPIH YCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEEWIRSVNFTDNAKTIIVQL NTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWNNTLKQIASKLREQFGNNKTII

FKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQIINMWQKVGK AMYAPPISGQIRCSSNITGLLLTRDGGNSNNESEIFRPGGGDMRDNWRSELYKYKWKIEPLGVAPTKAKRR WQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQL QARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNHTTWMEWDREINNYTSLIHSLIEE SQNQQEKNEQELLELDKWASLWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTH

LPTPRGPDRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEA LKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEWQGACRAIRHIPRRIRQGLERILL (SEQ ID NO: 26; GENBANK® Accession No. K03455, incorporated by reference herein as present in the database on May 4, 2015).

IgA: A polypeptide belonging to the class of antibodies that are substantially encoded by a recognized immunoglobulin alpha gene. In humans, this class or isotype comprises IgAi and IgA2. IgA antibodies can exist as monomers, polymers (referred to as plgA) of predominantly dimeric form, and secretory IgA. The constant chain of wild-type IgA contains an 18-amino-acid extension at its C-terminus called the tail piece (tp). Polymeric IgA is secreted by plasma cells with a 15-kDa peptide called the J chain linking two monomers of IgA through the conserved cysteine residue in the tail piece.

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IgG: A polypeptide belonging to the class or isotype of antibodies that are substantially encoded by a recognized immunoglobulin gamma gene. In humans, this class comprises IgGi, IgG2, IgG3, and IgG₄. In mice, this class comprises IgGi, IgG2a, IgG2b, IgG3.

Immune complex: The binding of antibody or antigen binding fragment (such as a scFv) to a soluble antigen forms an immune complex. The formation of an immune complex can be detected through conventional methods known to the skilled artisan, for instance immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (for example, Western blot), magnetic resonance imaging, CT scans, X-ray and affinity chromatography. Immunological binding properties of selected antibodies may be quantified using methods well known in the art.

Isolated: A biological component (such as a nucleic acid, peptide, protein or protein complex, for example an antibody) that has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, that is, other chromosomal and extra-chromosomal DNA and RNA, and proteins. Thus, isolated nucleic acids, peptides and proteins include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell, as well as, chemically synthesized nucleic acids. A isolated nucleic acid, peptide or protein, for example an antibody, can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.

Linker: A bi-functional molecule that can be used to link two molecules into one contiguous molecule, for example, to link an effector molecule to an antibody. In some embodiments, the provided conjugates include a linker between the effector molecule or detectable marker and an antibody. In some cases, a linker is a peptide within an antigen binding fragment (such as an Fv fragment) which serves to indirectly bond the Vh and Vl. Non-limiting examples of peptide linkers include a (G₄S)i linker, a (G₄S)2 linker, or a (G₄S)3 linker.

The terms “conjugating,” “joining,” “bonding,” or “linking” can refer to making two molecules into one contiguous molecule; for example, linking two polypeptides into one contiguous polypeptide, or covalently attaching an effector molecule or detectable marker radionuclide or other molecule to a polypeptide, such as an scFv. In the specific context, the terms include reference to joining a ligand, such as an antibody moiety, to an effector molecule. The linkage can be either by chemical or recombinant means. “Chemical means” refers to a reaction between the antibody moiety and the effector molecule such that there is a covalent bond formed between the two molecules to form one molecule.

Nucleic acid molecule: A polymeric form of nucleotides, which may include both sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide. The term “nucleic acid molecule” as used herein is synonymous with “nucleic acid” and “polynucleotide.” A nucleic acid molecule is usually at least 10 bases in length, unless otherwise specified. The term includes

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PCT/US2016/060390 single- and double-stranded forms of DNA. A polynucleotide may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages. “cDNA” refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form. “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.

Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter, such as the CMV promoter, is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington's Pharmaceutical Science, 22th ed., Pharmaceutical Press, London, UK (2012), describes compositions and formulations suitable for pharmaceutical delivery of the disclosed agents.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers comprises, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, added preservatives (such as on-natural preservatives), and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In particular examples, the pharmaceutically acceptable carrier is sterile and suitable for parenteral administration to a subject for example, by injection. In some embodiments, the active agent and pharmaceutically acceptable carrier are provided in a unit dosage form such as a pill or in a selected quantity in a vial. Unit dosage forms comprises one dosage or multiple dosages (for example, in a vial from which metered dosages of the agents can selectively be dispensed).

Polypeptide: A polymer in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the Doptical isomer can be used, the L-isomers being preferred. The terms “polypeptide” or “protein” as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. A polypeptide includes both naturally occurring proteins, as well as those that are recombinantly or synthetically produced. A polypeptide has an amino terminal (N-terminal) end and a carboxy-terminal end. In some embodiments, the polypeptide is a disclosed antibody or a fragment thereof.

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Polypeptide modifications: polypeptides can be modified by a variety of chemical techniques to produce derivatives having essentially the same activity and conformation as the unmodified peptides, and optionally having other desirable properties. For example, carboxylic acid groups of the protein, whether carboxyl-terminal or side chain, may be provided in the form of a salt of a pharmaceutically-acceptable cation or esterified to form a C1-C16 ester, or converted to an amide of formula NR4R2 wherein Ri and R2 are each independently H or C1-C16 alkyl, or combined to form a heterocyclic ring, such as a 5- or 6- membered ring. Amino groups of the peptide, whether amino-terminal or side chain, may be in the form of a pharmaceutically-acceptable acid addition salt, such as the HC1, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or may be modified to C1-C16 alkyl or dialkyl amino or further converted to an amide.

Hydroxyl groups of the peptide side chains can be converted to C1-C16 alkoxy or to a C1-C16 ester using well-recognized techniques. Phenyl and phenolic rings of the peptide side chains can be substituted with one or more halogen atoms, such as F, Cl, Br or I, or with C1-C16 alkyl, C1-C16 alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids. Methylene groups of the peptide side chains can be extended to homologous C2-C4 alkylenes. Thiols can be protected with any one of a number of wellrecognized protecting groups, such as acetamide groups.

Recombinant: A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. A recombinant protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. In several embodiments, a recombinant protein is encoded by a heterologous (for example, recombinant) nucleic acid that has been introduced into a host cell, such as a bacterial or eukaryotic cell. The nucleic acid can be introduced, for example, on an expression vector having signals capable of expressing the protein encoded by the introduced nucleic acid or the nucleic acid can be integrated into the host cell chromosome.

Sequence identity: The similarity between amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet etal., Nucleic Acids Research 16.T0881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988.

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Altschul et al., Nature Genet. 6:119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul etal., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.

Homo logs and variants of a Vl or a Vh of an antibody that specifically binds a polypeptide are typically characterized by possession of at least about 75%, for example at least about 80%, 85%, 90%,

91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full length alignment with the amino acid sequence of interest. Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.

Terms used to describe sequence relationships between two or more nucleotide sequences or amino acid sequences include “reference sequence,” “selected from,” “comparison window,” “identical,” “percentage of sequence identity,” “substantially identical,” “complementary,” and “substantially complementary.”

For sequence comparison of nucleic acid sequences, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters are used. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970, by the search for similarity method of Pearson & Lipman, Proc. Nat’l. Acad. Sci. USA 85:2444, 1988, by computerized implementations of these algorithms (GAP, BESTF1T, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4^th ed, Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, lohn Wiley & Sons, New York, through supplement 104, 2013). One example of a useful

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PCT/US2016/060390 algorithm is PILEUP. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360, 1987. The method used is similar to the method described by Higgins & Sharp, CABIOS 5:151-153, 1989. Using PILEUP, a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al., Nuc. Acids Res. 12:387395, 1984.

Another example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and the BLAST 2.0 algorithm, which are described in Altschul et al., J. Mol. Biol. 215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402, 1977. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLASTP program (for amino acid sequences) uses as defaults a word length (W) of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989). An oligonucleotide is a linear polynucleotide sequence of up to about 100 nucleotide bases in length.

Specifically bind: When referring to an antibody or antigen binding fragment, refers to a binding reaction which determines the presence of a target protein, peptide, or polysaccharide in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated conditions, an antibody binds preferentially to a particular target protein, peptide or polysaccharide (such as an antigen present on the surface of a pathogen, for example HIV-1 Env) and does not bind in a significant amount to other proteins or polysaccharides present in the sample or subject. Specific binding can be determined by methods known in the art. With reference to an antibody-antigen complex, specific binding of the antigen and antibody has a Kd of less than about 10⁷ Molar, such as less than about 10⁸ Molar, 10⁹, or even less than about IO¹⁰ Molar.

Kd refers to the dissociation constant for a given interaction, such as a polypeptide ligand interaction or an antibody antigen interaction. For example, for the bimolecular interaction of an antibody or antigen binding fragment and an antigen it is the concentration of the individual components of the bimolecular interaction divided by the concentration of the complex.

The antibodies disclosed herein specifically bind to a defined target (or multiple targets, in the case of a bispecific antibody). Thus, an antibody that specifically binds to an epitope on gp41 is an antibody that binds substantially to gp41, including cells or tissue expressing gp41, substrate to which the gp41 is attached, or gp41 in a biological specimen. It is, of course, recognized that a certain degree of non-specific interaction may occur between an antibody or conjugate including an antibody (such as an antibody that specifically binds gp41 or conjugate including such antibody) and a non-target (such as a cell that does not express gp41). Typically, specific binding results in a much stronger association between the antibody and

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PCT/US2016/060390 protein or cells bearing the antigen than between the antibody and protein or cells lacking the antigen. Specific binding typically results in greater than 2-fold, such as greater than 5-fold, greater than 10-fold, or greater than 100-fold increase in amount of bound antibody (per unit time) to a protein including the epitope or cell or tissue expressing the target epitope as compared to a protein or cell or tissue lacking this epitope. Specific binding to a protein under such conditions requires an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats are appropriate for selecting antibodies or other ligands specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow & Lane, Antibodies, A Laboratory Manual, 2^nd ed., Cold Spring Harbor Publications, New York (2013), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

Subject: Living multi-cellular vertebrate organisms, a category that includes human and nonhuman mammals. In an example, a subject is a human. In a particular example, the subject is a newborn infant. In an additional example, a subject is selected that is in need of inhibiting of an HIV-1 infection. For example, the subject is either uninfected and at risk of HIV-1 infection or is infected in need of treatment.

Therapeutically effective amount: The amount of agent, such as a disclosed gp41 specific antibody or antigen binding fragment that is sufficient to prevent, treat (including prophylaxis), reduce and/or ameliorate the symptoms and/or underlying causes of a disorder or disease, for example to prevent, inhibit, and/or treat HIV-1 infection. In some embodiments, a therapeutically effective amount is sufficient to reduce or eliminate a symptom of a disease, such as AIDS. For instance, this can be the amount necessary to inhibit or prevent HIV-1 replication or to measurably alter outward symptoms of the HIV-1 infection. In general, this amount will be sufficient to measurably inhibit HIV-1 replication or infectivity. Ideally, a therapeutically effective amount provides a therapeutic effect without causing a substantial cytotoxic effect in the subject.

In some embodiments, administration of a therapeutically effective amount of a disclosed antibody or antigen binding fragment that binds to gp41 can reduce or inhibit an HIV-1 infection (for example, as measured by infection of cells, or by number or percentage of subjects infected by HIV-1, or by an increase in the survival time of infected subjects) by a desired amount, for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable HIV-1 infection), as compared to a suitable control.

Several preparations disclosed herein are administered in therapeutically effective amounts. A therapeutically effective amount of an antibody or antigen binding fragment that specifically binds gp41 that is administered to a subject will vary depending upon a number of factors associated with that subject, for example the overall health and/or weight of the subject. A therapeutically effective amount can be determined by varying the dosage and measuring the resulting therapeutic response, such as, for example, a reduction in viral titer. Therapeutically effective amounts also can be determined through various in vitro, in vivo or in situ immunoassays.

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A therapeutically effective amount encompasses a fractional dose that contributes in combination with previous or subsequent administrations to attaining a therapeutic response. For example, a therapeutically effective amount of an agent can be administered in a single dose, or in several doses, for example daily, during a course of treatment lasting several days or weeks. However, the therapeutically effective amount can depend on the subject being treated, the severity and type of the condition being treated, and the manner of administration. A unit dosage form of the agent can be packaged in a therapeutic amount, or in multiples of the therapeutic amount, for example, in a vial (e.g., with a pierceable lid) or syringe having sterile components.

Transformed: A transformed cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the term transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of DNA by electroporation, lipofection, and particle gun acceleration.

Treating or preventing a disease: Preventing a disease refers to inhibiting the full development of a disease or condition, for example, in a subject who is at risk of or has an HIV-1 infection. Treating a disease refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. The term “ameliorating,” with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the viral load, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease for the purpose of reducing the risk of developing pathology.

The term “prevents” does not necessarily mean that an agent completely eliminates the disease or condition, so long as at least one characteristic of the disease or condition is eliminated. Thus, an antibody that inhibits or prevents an infection, can, but does not necessarily completely, eliminate such an infection, so long as the infection is measurably diminished, for example, by at least about 50%, such as by at least about 70%, or about 80%, or even by about 90% the infection in the absence of the agent, or in comparison to a reference agent.

Vector: A vector comprises nucleic acid sequences (for example, mRNA or DNA) that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known in the art. Viral vectors are recombinant nucleic acid vectors having at least some nucleic acid sequences derived from one or more viruses. In some embodiments, a viral vector is provided that comprises one or more nucleic acid molecules encoding a disclosed antibody or antigen binding fragment that specifically binds to HIV-1 gp41 and neutralizes HIV-1. In some embodiments, the viral vector can be an adeno-associated virus (AAV) vector. A replication

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PCT/US2016/060390 deficient viral vector is a vector that requires complementation of one or more regions of the viral genome required for replication due to a deficiency in at least one replication-essential gene function. For example, such that the viral vector does not replicate in typical host cells, especially those in a human patient that could be infected by the viral vector in the course of a therapeutic method.

VRCOl-class antibody, heavy chain, or light chain: A class of antibodies that bind to the CD4 binding site on gpl20 and can neutralize HIV-1 infection, as well as heavy and light chains thereof. The prototypical member of the VRCOl-class of antibodies - VRC01 - can neutralize over 90% of circulating HIV-1 isolates with an average 50% inhibitory concentration (IC50) of ~0.3 Ug/ml. Despite overall sequence differences between VRCOl-class antibodies, antibody-gpl20 co-crystal structures revealed VRCOl-class recognition of gpl20 to be consistent across the class. Indeed, three-dimensional structure analysis of HIV-1 gpl20 from different HIV-1 clades in complexes with different VRCOl-class antibodies from multiple donors show that the VRCOl-class antibodies share striking similarity in physical structure, and revealed several antibody features that contribute to gpl20 binding and HIV-1 neutralization. The substantial structural and ontogenetic characterization of VRCOl-class of antibodies allows recognition of the members of this class by interrogation of antibody sequence.

For example, the Vh of a VRCOl-class antibody has a VH1-2 germline origin, wherein the VRCOlclass Vh encoding sequence is from 20-35% (such as 25-30%) divergent from the corresponding germline gene sequence. The VRCOl-class Vh includes a tryptophan residue at kabat position 50 (Vh Trpso), an asparagine residue at kabat position 58 (Vh Asms), and an arginine residue at kabat position 71 (Vh Arg7i). These residues form specific interactions with amino acids on gpl20 that contribute to the VRCOl-class specificity and neutralization properties. When a VRCOl-class antibody is bound to gpl20, Vh Trpso forms a hydrogen bond with gpl20 Asn₂so, Vh Asps_x forms hydrogen bonds with gpl20 Arg456 and Gly458, Vh Arg7i forms salt bridges with gpl20 Asp368, and Vh TrplOOB forms a hydrogen bond with gpl20 Asn₂79Further, the V_L of a VRCOl-class antibody has an IGKV1-33, IGKV3-11, IGKV3-15, IGKV3-20, or IGFV2-14 germline origin, wherein the VRCOl-class Vl encoding sequence is from 15-35% (such as 2530%) divergent from the corresponding germline gene sequence. The VRCOl-class Vl includes either a FCDR1 (kabat positioning) with a 2-6 amino acid deletion, or a FCDR1 with glycine residues at kabat positions 28 and 30. The deletion or the presence of the glycine residues provides flexibility that allows the FCDR1 to avoid structural clash with the D loop of gpl20 when the antibody is bound to the CD4 binding site. Further, the VRCOl-class Vl includes an FCDR3 that is five amino acids in length (according to kabat positioning) and includes a hydrophobic residue (such as leucine or tyrosine) at kabat position 91, deletion of kabat positions 92-95, and a glutamate or glutamine residue at kabat position 96. The hydrophobic residue at position 91 packs against the backbone of gpl20 Foop D, and the glutamate or glutamine residue at kabat position 96 interacts with a conserved electropositive region on the base of the gpl20 V5 domain.

Non-limiting examples of antibodies that fall within the VRCOl-class include the VRC01, VRC03,

VRC07, VRC07-523, VRC13, 3BCN117, 12A12, 12A21, VRC-PG04, NIH45-46, VRC23, VRC-CH30,

VRC-CH31, and VRC-PG20 antibodies. Description, characterization, and productions of these antibodies,

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PCT/US2016/060390 as well as the VRC01-class of antibodies is available and familiar to the person of ordinary skill in the art (see, e.g., Diskin etal., Science, 334(6060):1289-93, 2011; Kwong and Mascola, Immunity, 37, 412-425, 2012; Li et al., J. Virol., 85, 8954-8967, 2011; Rudicell et al., J. Virol., 88, 12669-12682, 2012; Scheid et al., Science, 333(6049):1633-1637, 2011; West etal., PNAS, 109:E2083-2090, 2012; Wu etal., Science, 329(5993):856-861, 2010; Wu et al., Science, 333(6049):1593-1602, 2011; Zhou et al., Immunity, 39:245258, 2013; Georgiev etal., Science, 340:751-756, 2013; Zhu etal., PNAS, 110, E4088-E4097, 2013; and WIPO Pub. Nos. WO 2012/158948, WO2011038290, WO2012154312, WO2013142324 and WO2013016468, each of which is incorporated by reference herein in its entirety).

The VRC01, VRC07, VRC07-523 (VRC07H-G54H heavy and VRC01L-ElI2del-V3S light), and VRC07-544 (VRC07H-I37V/G54H/T93A heavy and VRC01-EIdel-V3S light) antibodies are described in U.S. Pat. Pubs. 8337036 and 2014/0322163 and Rudicell et al., J. Virol., 88:12669-12682, 2014, each of which is incorporated by reference in its entirety. The Vh and Vl sequences of these antibodies are provided below:

VRC01 V_H

QVQLVQSGGQMKKPGESMRISCRASGYEFIDCTLNWIRLAPGKRPEWMGWLKPRGGAVNYARPLQGRVTMTR DVYSDTAFLELRSLTVDDTAVYFCTRGKNCDYNWDFEHWGRGTPVIVSS (SEQ ID NO: 27)

VRC01 V_L

EIVLTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRLVIYSGSTRAAGIPDRFSGSRWGPDYNLT ISNLESGDFGVYYCQQYEFFGQGTKVQVDIK (SEQ ID NO: 28)

VRC07 V_H

QVRLSQSGGQMKKPGDSMRISCRASGYEFINCPINWIRLAPGKRPEWMGWMKPRGGAVSYARQLQGRVTMTR DMYSETAFLELRSLTSDDTAVYFCTRGKYCTARDYYNWDFEHWGQGTPVTVSS (SEQ ID NO: 29)

VRC07 V_L

VRC07-523 V_H

QVRLSQSGGQMKKPGDSMRISCRASGYEFINCPINWIRLAPGKRPEWMGWMKPRHGAVSYARQLQGRVTMTR DMYSETAFLELRSLTSDDTAVYFCTRGKYCTARDYYNWDFEHWGQGTPVTVSS (SEQ ID NO: 30)

VRC07-523 V_L

SLTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRLVIYSGSTRAAGIPDRFSGSRWGPDYNLTIS NLESGDFGVYYCQQYEFFGQGTKVQVDIK (SEQ ID NO: 31)

VRC07-544 V_H

QVRLSQSGGQMKKPGDSMRISCRASGYEFINCPINWVRLAPGKRPEWMGWMKPRHGAVSYARQLQGRVTMTR DMYSETAFLELRSLTSDDTAVYFCARGKYCTARDYYNWDFEHWGQGTPVTVSS (SEQ ID NO: 32)

VRC07-544 V_L

SLTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRLVIYSGSTRAAGIPDRFSGSRWGPDYNLTIS

NLESGDFGVYYCQQYEFFGQGTKVQVDIK (SEQ ID NO: 33)

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II. Description of Several Embodiments

Isolated monoclonal antibodies and antigen binding fragments that specifically bind an epitope on gp41 are provided. The antibodies and antigen binding fragments can be fully human. In several embodiments, the antibodies and antigen binding fragments can be used to neutralize HIV-1 infection. Also disclosed herein are compositions including the antibodies and antigen binding fragments and a pharmaceutically acceptable carrier. Nucleic acids encoding the antibodies or antigen binding fragments, expression vectors (such as adeno-associated virus (AAV) viral vectors) including these nucleic acids are also provided.

The antibodies, antigen binding fragments, nucleic acid molecules, expression vectors, and compositions can be used for research, diagnostic and therapeutic purposes. For example, the monoclonal antibodies and antigen binding fragments can be used to diagnose or treat a subject with an HIV-1 infection, or can be administered prophylactically to prevent HIV-1 infection in a subject. In some embodiments, the antibodies can be used to determine HIV-1 titer in a subject.

A. Neutralizing Monoclonal Antibodies and Antigen Binding Fragments

The parent 10E8 antibody neutralizes 98% of a panel of 200 HIV-1 pseudotyped viruses with an IC50 of less than 50 Ug/ml (see Example 1). However, as disclosed in Example 1, the solubility of the 10E8 antibody may not be optimal for commercial production purposes. Earlier attempts at modifying the 10E8 antibody successfully improved the solubility and potency of the antibody, but caused an increase in autoreactivity. In some embodiments, disclosed herein are modified 10E8 antibodies and antigen binding fragments having the CDRs of the 10E8 antibody and framework region amino acid substitutions compared to the parent 10E8 antibody sequence that impart an improved combination of neutralization, solubility, and auto-reactivity properties relative to 10E8. In additional embodiments, the modified 10E8 antibodies include amino acid substitutions in the CDRs that impart an improved combination of neutralization, solubility, and auto-reactivity properties relative to 10E8.

The discussion of monoclonal antibodies below refers to monoclonal antibodies that include a Vh and a Vl including CDRs with reference to the IMGT numbering scheme (unless the context indicates otherwise). The person of ordinary skill in the art will understand that various CDR numbering schemes (such as the Rabat, Chothia or IMGT numbering schemes) can be used to determine CDR positions. The amino acid sequence and the CDR positions of the heavy and light chains of the 10E8 antibody, and several variants thereof, according to the IMGT numbering scheme are shown in Table 1.

Table 1. IMGT CDR sequences of 10E8 and 10E8 variants.

10E8 V_H
V_h	SEQ ID NO: 1 positions (linear)	CDR protein sequence	HCDR SEQ ID NO
HCDR1	26-33	GFDFDNAW	7
HCDR2	51-60	ITGPGEGWSV	8
HCDR3	99-120	ARTGKYYDFWSGYPPGEEYFQD	9

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10E8 Vl
V_l	SEQ ID NO: 2 positions (linear)	A.A. Sequence	LCDR SEQ ID NO
LCDR1	26-31	SLRSHY	10
LCDR2	49-51	GKN	11
LCDR3	88-99	SSRDKSGSRLSV	12
10E8v4 SlOOcW or 10E8v5 SlOOcW V_H
V_H	SEQ ID NO: 35 (v4) or SEQ ID NO: 38 (v5) positions (linear)	CDR protein sequence	HCDR SEQ ID NO
HCDR1	26-33	GFDFDNAW	7
HCDR2	51-60	ITGPGEGWSV	8
HCDR3	99-120	ARTGKYYDFWWGYPPGEEYFQD	52
10E8v4 DS or 10E8v5 DS V_H
V_H	SEQ ID NO: 36 (v4) or SEQ ID NO: 39 (v5) positions (linear)	CDR protein sequence	HCDR SEQ ID NO
HCDR1	26-33	GFDFDNAW	7
HCDR2	51-60	ITGPGEGWSV	8
HCDR3	99-120	ARTGKYYDFWSGCPPGEEYFQD	53
10E8v4 SlOOcW-DS or 10E8v5 SlOOcW-DS V_H
V_H	SEQ ID NO: 37 (v4) or SEQ ID NO: 40 (v5) positions (linear)	CDR protein sequence	HCDR SEQ ID NO
HCDR1	26-33	GFDFDNAW	7
HCDR2	51-60	ITGPGEGWSV	8
HCDR3	99-120	ARTGKYYDFWWGCPPGEEYFQD	54
10E8v4 DS or 10E8v5 DS V_L
V_L	SEQ ID NO: 41 positions (linear)	A.A. Sequence	LCDR SEQ ID NO
LCDR1	26-31	SLRCHY	55
LCDR2	49-51	GKN	11
LCDR3	88-99	SSRDKSGSRLSV	12
10E8v4 Y32W or 10E8v5 Y32W Vl
V_l	SEQ ID NO: 42 positions (linear)	A.A. Sequence	LCDR SEQ ID NO
LCDR1	26-31	SLRSHW	56
LCDR2	49-51	GKN	11
LCDR3	88-99	SSRDKSGSRLSV	12
10E8v4 Y32F or 10E8v5 Y32F V_L
V_L	SEQ ID NO: 43 positions (linear)	A.A. Sequence	LCDR SEQ ID NO
LCDR1	26-31	SLRSHF	57
LCDR2	49-51	GKN	11
LCDR3	88-99	SSRDKSGSRLSV	12
10E8v4 DS Y32W or 10E8v5 DS Y32W Vl
V_l	SEQ ID NO: 58 positions (linear)	A.A. Sequence	LCDR SEQ ID NO
LCDR1	26-31	SLRCHW	85
LCDR2	49-51	GKN	11
LCDR3	88-99	SSRDKSGSRLSV	12
10E8v4 DS Y32F or 10E8v5 DS Y32F V_L
V_L	SEQ ID NO: 59 positions (linear)	A.A. Sequence	LCDR SEQ ID NO

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LCDR1	26-31	SLRCHF	86
LCDR2	49-51	GKN	11
LCDR3	88-99	SSRDKSGSRLSV	12

10E8 Variant 4 H6-51 l-4mut V_H (SEQ ID NO: 5) + rL3-6mut V_L (SEQ ID NO: 6)
V_H	SEQ ID NO: 5 positions	CDR protein sequence	HCDR SEQ ID NO
HCDR1	26-33	GFDFDNAW	7
HCDR2	51-60	ITGPGEGWSV	8
HCDR3	99-120	ARTGKYYDFWSGYPPGEEYFQD	9

V_L	SEQ ID NO: 2 positions (linear)	A.A. Sequence	LCDR SEQ ID NO
LCDR1	26-31	SLRSHY	10
LCDR2	49-51	GKN	11
LCDR3	88-99	SSRDKSGSRLSV	12

10E8 Variant 4 - SlOOcF H6-511-4mut-S100cF V_H (SEQ ID NO: 75) + rL3-6mut V_L (SEQ ID NO: 6)
V_H	SEQ ID NO: 75 positions	CDR protein sequence	HCDR SEQ ID NO
HCDR1	26-33	GFDFDNAW	7
HCDR2	51-60	ITGPGEGWSV	8
HCDR3	99-120	ARTGKYYDFWFGYPPGEEYFQD	77

V_L	SEQ ID NO: 6 positions	A.A. Sequence	LCDR SEQ ID NO
LCDR1	26-31	SLRSHY	10
LCDR2	49-51	GKN	11
LCDR3	88-99	SSRDKSGSRLSV	12

10E8 Variant 4 - V5R, SlOOcF H6-511-4mut V_H (SEQ ID NO: 76) + rL3-6mut V_L (SEQ ID NO: 6)
V_H	SEQ ID NO: 76 positions	CDR protein sequence	HCDR SEQ ID NO
HCDR1	26-33	GFDFDNAW	7
HCDR2	51-60	ITGPGEGWSV	8
HCDR3	99-120	ARTGKYYDFWFGYPPGEEYFQD	77

V_L	SEQ ID NO: 6 positions	A.A. Sequence	LCDR SEQ ID NO
LCDR1	26-31	SLRSHY	10
LCDR2	49-51	GKN	11
LCDR3	88-99	SSRDKSGSRLSV	12

10E8 variant 1 (10E8vl)

As disclosed in the examples, the “10E8 variant 1,” which includes the HC6-S74Y-511 Vh (SEQ ID

NO: 3) and rL3 Vl (SEQ ID NO: 4), specifically binds gp41 and has an unexpectedly superior combination of HIV-1 neutralization breadth and potency, solubility, and auto-reactivity. The HC6-S74Y-511 Vh

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10E8 light chain CDRs, as well as E7D, I45V, V58I, S76T, D83E, and E85D framework amino acid substitutions compared to the 10E8 Vl (SEQ ID NO: 2).

Accordingly, in some embodiments, a disclosed antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs (such as SEQ ID NOs: 7, 8, and 9) and can further include arginine, alanine, aspartate, tyrosine, lysine, threonine, threonine, and isoleucine residues at kabat positions 3, 5, 72, 74, 75, 77, 84, and 110, respectively, and a Vl including the 10E8 Vl CDRs (such as SEQ ID NOs: 10, 11, 12) and aspartate, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 7, 45, 58, 76, 83, and 85, respectively.

In additional embodiments, the antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs (such as SEQ ID NOs: 7, 8, and 9), arginine, alanine, aspartate, tyrosine, lysine, threonine, threonine, and isoleucine residues at kabat positions 3, 5, 72, 74, 75, 77, 84, and 110, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 3 (HC6S74Y-511), and a Vl including the 10E8 Vl CDRs (such as SEQ ID NOs: 10, 11, and 12), and aspartate, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 7, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 4 (rL3).

In more embodiments, the antibody or antigen binding fragment comprises a Vh including the amino acid sequence set forth as SEQ ID NO: 3 (HC6-S74Y-511), and a Vl including the amino acid sequence set forth as SEQ ID NO: 4 (rL3).

10E8 variant 3 (10E8v3)

As disclosed in the examples, the “10E8 variant 3,” which includes the H6-511-4mut Vh (SEQ ID NO: 5) and rL3 Vl (SEQ ID NO: 4), specifically binds gp41 and has an unexpectedly superior combination of HIV-1 neutralization breadth and potency, solubility, and auto-reactivity. The H6-51 l-4mut Vh includes the 10E8 heavy chain CDRs, as well as Q3R, A61E, P62S, E64K, L72D, S74T, I75K, F77T, L82cV, M84T, S87T, L89Y, R105Q, ΤΙ 101 framework amino acid substitutions compared to the 10E8 Vh (SEQ ID NO: 1). The rL3 Vl is described above.

Accordingly, in some embodiments, a disclosed antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs (such as SEQ ID NOs: 7, 8, and 9) and arginine, glutamate, serine, lysine, aspartate, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively, and a Vl including the 10E8 Vl CDRs (such as SEQ ID NOs: 10, 11, 12) and aspartate, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 7, 45, 58, 76, 83, and 85, respectively.

In additional embodiments, the antibody or antigen binding fragment comprises a Vh including the

10E8 Vh CDRs (such as SEQ ID NOs: 7, 8, and 9) and arginine, glutamate, serine, lysine, aspartate,

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In more embodiments, the antibody or antigen binding fragment comprises a Vh including the amino acid sequence set forth as SEQ ID NO: 5 (H6-51 l-4mut), and a Vl including the amino acid sequence set forth as SEQ ID NO: 4 (rL3).

10E8 variant 4 (10E8v4)

As disclosed in the examples, the “10E8 variant 4,” which includes the H6-511-4mut Vh (SEQ ID NO: 5) and rL3-6mut Vl (SEQ ID NO: 6), specifically binds gp41 and has an unexpectedly superior combination of HIV -1 neutralization breadth and potency, solubility, and auto-reactivity. The H6-511 -4mut Vh is described above. The rL3-6mut Vl includes the 10E8 light chain CDRs, as well as SI A, Y2S, E7D, T8P, G9A, G16K, R17Q, I45V, V58I, S76T, D83E, E85D framework amino acid substitutions compared to the 10E8 V_L (SEQ ID NO: 2).

Accordingly, in some embodiments, a disclosed antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs (such as SEQ ID NOs: 7, 8, and 9) and arginine, glutamate, serine, lysine, aspartate, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively, and a Vl including the 10E8 Vl CDRs (such as SEQ ID NOs: 10, 11, 12) and alanine, serine, aspartate, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively

In additional embodiments, the antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs (such as SEQ ID NOs: 7, 8, and 9) and arginine, glutamate, serine, lysine, aspartate, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 5 (H6-51 l-4mut), and a Vl including the 10E8 Vl CDRs (such as SEQ ID NOs: 10, 11, and 12), alanine, serine, aspartate, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 6 (rL3-6mut).

In more embodiments, the antibody or antigen binding fragment comprises a Vh including the amino acid sequence set forth as SEQ ID NO: 5 (H6-51 l-4mut), and a Vl including the amino acid sequence set forth as SEQ ID NO: 6 (rL3-6mut).

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10E8 variants (10E8v5)

As disclosed in the examples, the “10E8 variant 5,” which includes the HC6-S74Y-511 Vh (SEQ ID NO: 3) and rL3-6mut Vl (SEQ ID NO: 6), specifically binds gp41 and has an unexpectedly superior combination of HIV-1 neutralization breadth and potency, solubility, and auto-reactivity. The HC6-S74Y511 Vh and rL3-6mut Vl are described above.

Accordingly, in some embodiments, a disclosed antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs (such as SEQ ID NOs: 7, 8, and 9) and can further include arginine, alanine, aspartate, tyrosine, lysine, threonine, threonine, and isoleucine residues at kabat positions 3, 5, 72, 74, 75, 77, 84, and 110, respectively, and a Vl including the 10E8 Vl CDRs (such as SEQ ID NOs: 10, 11, 12) and alanine, serine, aspartate, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively

In additional embodiments, the antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs (such as SEQ ID NOs: 7, 8, and 9), arginine, alanine, aspartate, tyrosine, lysine, threonine, threonine, and isoleucine residues at kabat positions 3, 5, 72, 74, 75, 77, 84, and 110, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 3 (HC6S74Y-511), and a Vl including the 10E8 Vl CDRs (such as SEQ ID NOs: 10, 11, and 12), alanine, serine, aspartate, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 6 (rL3-6mut).

In more embodiments, the antibody or antigen binding fragment comprises a Vh including the amino acid sequence set forth as SEQ ID NO: 3 (HC6-S74Y-511), and a Vl including the amino acid sequence set forth as SEQ ID NO: 6 (rL3-6mut).

SlOOcW, SlOOcF, and V5R

As disclosed in the examples, incorporation of a hydrophobic amino acid substitution at VH kabat position SI00c (such as a SlOOcW or SlOOcF substitution) into certain 10E8 antibodies (such as the 10E8 variant 4 antibody) produces an antibody that specifically binds gp41 and has an unexpectedly superior combination of HIV-1 neutralization breadth and potency, solubility, and auto-reactivity. Accordingly, in some embodiments, any of the antibodies described herein (such as 10E8, or 10E8 variants including variant 1, 3, 4, or 5) can further include a SlOOcW substitution. In some embodiments, any of the antibodies described herein (such as 10E8, or 10E8 variants including variant 1, 3, 4, or 5) can further include a SlOOcF substitution.

10E8 variant 4 SlOOcF (10E8v4 SlOOcF)

In some embodiments, the antibody or antigen binding fragment comprises the Vh and Vl of the

10E8v4 antibody (VH: H6-511-4mut, SEQ ID NO: 5; VL: rL3-6mut, SEQ ID NO: 6) with a SlOOcF substitution in the Vh- Antibodies including such a Vh and Vl are denoted as “10E8v4 SlOOcF,” and

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PCT/US2016/060390 corresponding Vh and Vl amino acid sequences are provided herein as SEQ ID NOs: 75 and 6 respectively.

The 10E8v4 SlOOcF antibody specifically binds gp41 and neutralizes HIV-1, and also has an unexpectedly superior combination of HIV-1 neutralization breadth and potency, solubility, and auto-reactivity.

In some embodiments, the antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs with SlOOcF substitution (such as SEQ ID NOs: 7, 8, and 77) a Vl including the 10E8 Vl CDRs (such as SEQ ID NOs: 10, 11, 12), wherein the antibody specifically binds to gp41 and neutralizes HIV-1.

In some embodiments, the antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs with SlOOcF substitution (such as SEQ ID NOs: 7, 8, and 77) and arginine, glutamate, serine, lysine, aspartate, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively, and a Vl including the 10E8 Vl CDRs (such as SEQ ID NOs: 10, 11, 12) and alanine, serine, aspartate, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, wherein the monoclonal antibody specifically binds to gp41 and neutralizes HIV-1.

In additional embodiments, the antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs with SlOOcF substitution (such as SEQ ID NOs: 7, 8, and 77), arginine, glutamate, serine, lysine, aspartate, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 75 (H6-51 l-4mut-SlOOcF), and a V_L including the 10E8 V_L CDRs (such as SEQ ID NOs: 10, 11, and 12), alanine, serine, aspartate, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 6 (rL3-6mut), wherein the monoclonal antibody specifically binds to gp41 and neutralizes HIV-1.

In more embodiments, the antibody or antigen binding fragment comprises a Vh including the amino acid sequence set forth as SEQ ID NO: 75 (H6-511-4mut-S100cW), and a Vl including the amino acid sequence set forth as SEQ ID NO: 6 (rL3-6mut).

10E8 variant 4 VSR SlOOcF (10E8v4 V5R SlOOcF)

In some embodiments, the antibody or antigen binding fragment comprises the Vh and Vl of the 10E8v4 antibody (VH: H6-511-4mut, SEQ ID NO: 5; VL: rL3-6mut, SEQ ID NO: 6) with V5R and SlOOcF substitutions in the Vh. Antibodies including such a Vh and Vl are denoted as “10E8v4 V5R SlOOcF,” and corresponding Vh and Vl amino acid sequences are provided herein as SEQ ID NOs: 76 and 6 respectively. The 10E8v4 V5R SlOOcF antibody specifically binds gp41 and neutralizes HIV-1, and also has an unexpectedly superior combination of HIV-1 neutralization breadth and potency, solubility, and autoreactivity.

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In some embodiments, the antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs with SlOOcF substitution (such as SEQ ID NOs: 7, 8, and 77) and arginine, arginine glutamate, serine, lysine, aspartate, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 5, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively, and a Vl including the 10E8 Vl CDRs (such as SEQ ID NOs: 10, 11, 12) and alanine, serine, aspartate, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, wherein the monoclonal antibody specifically binds to gp41 and neutralizes HIV-1.

In additional embodiments, the antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs with SlOOcF substitution (such as SEQ ID NOs: 7, 8, and 77), arginine, arginine, glutamate, serine, lysine, aspartate, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 5, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 76 (H6-51 l-4mut-V5R SlOOcF), and a V_L including the 10E8 V_L CDRs (such as SEQ ID NOs: 10, 11, and 12), alanine, serine, aspartate, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 6 (rL3-6mut), wherein the monoclonal antibody specifically binds to gp41 and neutralizes HIV-1.

In more embodiments, the antibody or antigen binding fragment comprises a Vh including the amino acid sequence set forth as SEQ ID NO: 76 (H6-511-4mut-V5R SlOOcW), and a Vl including the amino acid sequence set forth as SEQ ID NO: 6 (rL3-6mut).

Additional modification of 10E8 variant 4 V5R SlOOcF

In some embodiments, the antibody or antigen binding fragment comprises the Vh and Vl of the 10E8v4 V5R SlOOcF antibody further comprising one or more amino acid substitutions that improve the HIV-1 neutralization breadth and potency, solubility, and/or auto-reactivity of the modified antibody compared to 10E8v4 V5R SlOOcF. The modified 10E8v4 V5R SlOOcF antibodies specifically bind gp41 and neutralize HIV-1, and also have an unexpectedly superior combination of HIV-1 neutralization breadth and potency, solubility, and auto-reactivity.

In some embodiments, the antibody or antigen binding fragment comprises a Vh including the amino acid sequence set forth as SEQ ID NO: 76 (H6-51 l-4mut-V5R SlOOcW) that further comprises one of a E1R, E1W, G15R, S25W, D28W, D30W, D30R, N31W, N31R, P52bR, D72R, or E81R substitution (kabat numbering) relative to SEQ ID NO: 76, and a Vl including the amino acid sequence set forth as SEQ ID NO: 6 (rL3-6mut), wherein the antibody specifically binds to gp41 and neutralizes HIV-1.

In some embodiments, the antibody or antigen binding fragment comprises a Vh including the amino acid sequence set forth as SEQ ID NO: 76 (H6-51 l-4mut-V5R SlOOcW), and a Vl including the amino acid sequence set forth as SEQ ID NO: 6 (rL3-6mut) that further comprises one of a D7W, A14R,

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Q17R, T18R, T20R, D26R, K51R, N53R, S65R, G68R, G77R, or G100R substitution (kabat numbering) relative to SEQ ID NO: 6, wherein the antibody specifically binds to gp41 and neutralizes HIV-1. In a preferred embodiment, the antibody or antigen binding fragment comprises a Vh including the amino acid sequence set forth as SEQ ID NO: 76 (H6-51 l-4mut-V5R SlOOcW), and a Vl including the amino acid sequence set forth as SEQ ID NO: 6 (rL3-6mut) that further comprises a G68R substitution (kabat numbering) relative to SEQ ID NO: 6, wherein the antibody specifically binds to gp41 and neutralizes HIV1.

In some embodiments, the antibody or antigen binding fragment comprises a Vh comprising the amino acid sequence set forth as SEQ ID NO: 76 (H6-51 l-4mut-V5R SlOOcW), and a Vl comprising the amino acid sequence set forth as SEQ ID NO: 6 (rL3-6mut), wherein the Vh and Vl further comprise one of the following pairs of substitution relative to SEQ ID NOs: 76 and 6, respectively: E1R and D7W; E1R and D26R; E1R and G68R; D28W and D7W; D28W and D26R; D28W and G68R; D28W and Q17R; D28W and T18R; D28W and T20R; D28W and K51R; D28W and G77R; P52bR and D7W; P52bR and D26R; P52bR and G68R; P52bR and Q17R; P52bR and T18R; P52bR and T20R; P52bR and K51R; P52bR and G77R; D72R and D7W; D72R and D26R; or D72R and G68R; and wherein the antibody specifically binds to gp41 and neutralizes HIV-1.

In some embodiments, the antibody or antigen binding fragment comprises a Vh comprising the amino acid sequence set forth as SEQ ID NO: 76 (H6-51 l-4mut-V5R SlOOcW), and a Vl comprising the amino acid sequence set forth as SEQ ID NO: 6 (rL3-6mut), wherein the Vh further comprises D72R, E1R D28W, and P52bR substitutions and wherein the antibody specifically binds to gp41 and neutralizes HIV-1. In some embodiments, the antibody or antigen binding fragment comprises a Vh comprising the amino acid sequence set forth as SEQ ID NO: 76 (H6-51 l-4mut-V5R SlOOcW), and a Vl comprising the amino acid sequence set forth as SEQ ID NO: 6 (rL3-6mut), wherein the Vl further comprises D7W, D26R, and G68R substitutions and wherein the antibody specifically binds to gp41 and neutralizes HIV-1. In some embodiments, the antibody or antigen binding fragment comprises a Vh comprising the amino acid sequence set forth as SEQ ID NO: 76 (H6-51 l-4mut-V5R SlOOcW), and a Vl comprising the amino acid sequence set forth as SEQ ID NO: 6 (rL3-6mut), wherein the Vh further comprises D72R, E1R D28W, and P52bR substitutions, and the Vl further comprises D7W, D26R, and G68R substitutions, and wherein the antibody specifically binds to gp41 and neutralizes HIV-1.

In some embodiments, the antibody or antigen binding fragment comprises a Vh comprising the amino acid sequence set forth as SEQ ID NO: 76 (H6-51 l-4mut-V5R SlOOcW), and a Vl comprising the amino acid sequence set forth as SEQ ID NO: 6 (rL3-6mut), wherein the Vh and/or Vl further comprise the amino acid substitutions of any one of the combinations of substitutions listed in Table 10 and wherein the antibody specifically binds to gp41 and neutralizes HIV-1. In some embodiments, the antibody or antigen binding fragment comprises a Vh comprising the amino acid sequence set forth as SEQ ID NO: 76 (H6-5114mut-V5R SlOOcW), and a Vl comprising the amino acid sequence set forth as SEQ ID NO: 6 (rL3-6mut), wherein the Vh and/or Vl further comprise the amino acid substitutions of listed in Table 10 for any one of

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PCT/US2016/060390 the 10E8-1053, 10E8-1054, 10E8-1055, 10E8-1056, 10E8-1057, 10E8-1058, 10E8-1059, 10E8-1060, 10E81061, 10E8-1062, 10E8-1063, 10E8-1064, 10E8-1065, 10E8-1066, 10E8-1067, 10E8-1068, 10E8-1069, 10E8-1070, 10E8-1071, 10E8-1072, 10E8-1073, 10E8-1074,10E8-1075, 10E8-1076, 10E8-1077, 10E81078, 10E8-1079, 10E8-1080, 10E8-1081, 10E8-1082, 10E8-1083, 10E8-1084, 10E8-1085, 10E8-1086, 10E8-1087, 10E8-1088, 10E8-1089, 10E8-1090, 10E8-1091, 10E8-1092, 10E8-1093, 10E8-1094, or 10E81095 antibodies, and wherein the antibody specifically binds to gp41 and neutralizes HIV-1.

In some embodiments, the antibody or antigen binding fragment comprises a Vh comprising the amino acid sequence set forth as SEQ ID NO: 76 (H6-51 l-4mut-V5R SlOOcW), and a Vl comprising the amino acid sequence set forth as SEQ ID NO: 6 (rL3-6mut) (corresponding to the 10E8-1073 antibody listed in Table 10), wherein the Vh further comprises a D28Y substitution and wherein the antibody specifically binds to gp41 and neutralizes HIV-1. In some embodiments, the antibody or antigen binding fragment comprises a Vh comprising the amino acid sequence set forth as SEQ ID NO: 76 (H6-51 l-4mut-V5R SlOOcW), and a Vl comprising the amino acid sequence set forth as SEQ ID NO: 6 (rL3-6mut), wherein the Vh further comprises a P52bR substitution, and the Vl further comprises D7W and G68R substitutions (corresponding to the 10E8-1081 antibody listed in Table 10), and wherein the antibody specifically binds to gp41 and neutralizes HIV-1.

10E8 variant 4 SlOOcW

In some embodiments, a disclosed antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs with SlOOcW substitution (such as SEQ ID NOs: 7, 8, and 52) and arginine, glutamate, serine, lysine, aspartate, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively, and a Vl including the 10E8 Vl CDRs (such as SEQ ID NOs: 10, If, 12) and alanine, serine, aspartate, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively

In additional embodiments, the antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs with SlOOcW substitution (such as SEQ ID NOs: 7, 8, and 52), arginine, glutamate, serine, lysine, aspartate, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 35 (H6-51 l-4mut-S100cW), and a V_L including the 10E8 V_L CDRs (such as SEQ ID NOs: 10, If, and 12), alanine, serine, aspartate, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 6 (rL3-6mut).

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In more embodiments, the antibody or antigen binding fragment comprises a Vh including the amino acid sequence set forth as SEQ ID NO: 35 (H6-511-4mut-S100cW), and a Vl including the amino acid sequence set forth as SEQ ID NO: 6 (rL3-6mut).

10E8 variant 5 SIOOcW

In some embodiments, a disclosed antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs with SIOOcW substitution (such as SEQ ID NOs: 7, 8, and 52) and can further include arginine, alanine, aspartate, tyrosine, lysine, threonine, threonine, and isoleucine residues at kabat positions 3, 5, 72, 74, 75, 77, 84, and 110, respectively, and a Vl including the 10E8 Vl CDRs (such as SEQ ID NOs: 10, 11, 12) and alanine, serine, aspartate, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively

In additional embodiments, the antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs with SIOOcW substitution (such as SEQ ID NOs: 7, 8, and 52), arginine, alanine, aspartate, tyrosine, lysine, threonine, threonine, and isoleucine residues at kabat positions 3, 5, 72, 74, 75, 77, 84, and 110, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 38 (HC6-S74Y-511-SIOOcW), and a V_L including the 10E8 V_L CDRs (such as SEQ ID NOs: 10, 11, and 12), alanine, serine, aspartate, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 6 (rL3-6mut).

In more embodiments, the antibody or antigen binding fragment comprises a Vh including the amino acid sequence set forth as SEQ ID NO: 38 (HC6-S74Y-511-SIOOcW), and a Vl including the amino acid sequence set forth as SEQ ID NO: 6 (rL3-6mut).

DS

As disclosed in the examples, incorporation of the “DS” modification, a non-native disulfide bond between the LCDR1 kabat position 30 and the HCDR3 kabat position lOOe, into certain 10E8 antibodies (such as the 10E8 variant 4 antibody) produces an antibody that specifically binds gp41 and has an unexpectedly superior combination of HIV-1 neutralization breadth and potency, solubility, and autoreactivity. Accordingly, any of the antibodies described herein (such as 10E8, or 10E8 variants including variant 1, 3, 4, or 5) can further include a DS substitution.

10E8 variant 4 DS

In some embodiments, a disclosed antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs with DS substitution (such as SEQ ID NOs: 7, 8, and 53) and arginine, glutamate, serine, lysine, aspartate, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110,

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In additional embodiments, the antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs with DS substitution (such as SEQ ID NOs: 7, 8, and 53), arginine, glutamate, serine, lysine, aspartate, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 36 (H651 l-4mut-DS), and a Vl including the 10E8 Vl CDRs with DS substitution (such as SEQ ID NOs: 55, 11, and 12), alanine, serine, aspartate, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 41 (rL3-6mut-DS).

In more embodiments, the antibody or antigen binding fragment comprises a Vh including the amino acid sequence set forth as SEQ ID NO: 36 (H6-511-4mut-DS), and a Vl including the amino acid sequence set forth as SEQ ID NO: 41 (rL3-6mut-DS).

10E8 variant 5 DS

In some embodiments, a disclosed antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs with DS substitution (such as SEQ ID NOs: 7, 8, and 53) and can further include arginine, alanine, aspartate, tyrosine, lysine, threonine, threonine, and isoleucine residues at kabat positions 3, 5, 72, 74, 75, 77, 84, and 110, respectively, and a Vl including the 10E8 Vl CDRs with DS substitution (such as SEQ ID NOs: 55, 11, 12) and alanine, serine, aspartate, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively

In additional embodiments, the antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs with DS substitution (such as SEQ ID NOs: 7, 8, and 53), arginine, alanine, aspartate, tyrosine, lysine, threonine, threonine, and isoleucine residues at kabat positions 3, 5, 72, 74, 75, 77, 84, and 110, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 39 (HC6-S74Y-511-DS), and a V_L including the 10E8 V_L CDRs with DS substitution (such as SEQ ID NOs: 55, 11, and 12), alanine, serine, aspartate, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 41 (rL3-6mut-DS).

In more embodiments, the antibody or antigen binding fragment comprises a Vh including the amino acid sequence set forth as SEQ ID NO: 39 (HC6-S74Y-511-DS), and a Vl including the amino acid sequence set forth as SEQ ID NO: 41 (rL3-6mut-DS).

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SlOOcW-DS

As disclosed in the examples, incorporation the SlOOcW and DS modifications, a non-native disulfide bond between the LCDR1 kabat position 30 and the HCDR3 kabat position lOOe, and a SlOOcW substitution, into certain 10E8 antibodies (such as the 10E8 variant 4 antibody) produces an antibody that specifically binds gp41 and has an unexpectedly superior combination of HIV-1 neutralization breadth and potency, solubility, and auto-reactivity. Accordingly, any of the antibodies described herein (such as 10E8, or 10E8 variants including variant 1, 3, 4, or 5) can further include SlOOcW and DS substitutions.

10E8 variant 4 SlOOcW-DS

In some embodiments, a disclosed antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs with slOOcW and DS substitutions (such as SEQ ID NOs: 7, 8, and 54) and arginine, glutamate, serine, lysine, aspartate, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and iso leucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively, and a Vl including the 10E8 Vl CDRs with DS substitution (such as SEQ ID NOs: 55, 11, 12) and alanine, serine, aspartate, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively

In additional embodiments, the antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs with S-lOOcW and DS substitutions (such as SEQ ID NOs: 7, 8, and 54), arginine, glutamate, serine, lysine, aspartate, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and iso leucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 37 (H6-51 l-4mut-S100cW-DS), and a V_L including the 10E8 V_L CDRs with DS substitution (such as SEQ ID NOs: 55, 11, and 12), alanine, serine, aspartate, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 41 (rL3-6mut-DS).

In more embodiments, the antibody or antigen binding fragment comprises a Vh including the amino acid sequence set forth as SEQ ID NO: 37 (H6-511-4mut-S100cW-DS), and a Vl including the amino acid sequence set forth as SEQ ID NO: 41 (rL3-6mut-DS).

10E8 variant 5 SlOOcW-DS

In some embodiments, a disclosed antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs with SlOOcW and DS substitutions (such as SEQ ID NOs: 7, 8, and 54) and can further include arginine, alanine, aspartate, tyrosine, lysine, threonine, threonine, and isoleucine residues at kabat positions 3, 5, 72, 74, 75, 77, 84, and 110, respectively, and a Vl including the 10E8 Vl CDRs with DS substitution (such as SEQ ID NOs: 55, 11, 12) and alanine, serine, aspartate, proline, alanine, lysine,

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17, 45, 58, 76, 83, and 85, respectively

10E8 Vh CDRs with SlOOcW and DS substitutions (such as SEQ ID NOs: 7, 8, and 54), arginine, alanine, aspartate, tyrosine, lysine, threonine, threonine, and isoleucine residues at kabat positions 3, 5, 72, 74, 75,

77, 84, and 110, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 40 (HC6-S74Y-511-S100cW-DS), and a V_L including the 10E8 V_L CDRs with DS substitution (such as SEQ ID NOs: 55, 11, and 12), alanine, serine, aspartate, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 41 (rL3-6mut-DS).

In more embodiments, the antibody or antigen binding fragment comprises a Vh including the amino acid sequence set forth as SEQ ID NO: 40 (HC6-S74Y-51 l-S100cW-DS), and a Vl including the amino acid sequence set forth as SEQ ID NO: 41 (rL3-6mut-DS).

Substitution at Vl kabat position 32

As disclosed in the examples, incorporation of an amino acid substitution at kabat position 32 of the Vl of certain 10E8 antibodies (such as the 10E8 variant 4 antibody) produces an antibody that specifically binds gp41 and has an unexpectedly superior combination of HfV-1 neutralization breadth and potency, solubility, and auto-reactivity. Accordingly, any of the antibodies described herein (such as 10E8, or 10E8 variants including variants 1, 3, 4, or 5; or 10E8 or 10E8 variants including variants 1, 3, 4, or 5 with a SlOOcW substitution; or 10E8 or 10E8 variants including variants 1, 3, 4, or 5 with a DS substitution; or 10E8 or 10E8 variants including variants 1, 3, 4, or 5 with SlOOcW-DS substitutions) can further include an amino acid substitution at kabat position 32 of the Vl. In some embodiments, the substation can be a Y32F substitution. In additional embodiments, the substitution can be a Y32W substitution.

In some embodiments, the Vl of any of the 10E8 variants disclosed in the “10E8 variant 4” section above can further include an amino acid substitution (such as a Y32F or Y32W substitution) at kabat position 32. In some embodiments, the Vl of any of the 10E8 variants disclosed in the “10E8 variant 5” section above can further include an amino acid substitution (such as a Y32F or Y32W substitution) at kabat position 32. In some embodiments, the Vl of any of the 10E8 variants disclosed in the “SlOOcW and SlOOcF’ section above can further include an amino acid substitution (such as a Y32F or Y32W substitution) at kabat position 32. In some embodiments, the Vl of any of the 10E8 variants disclosed in the “DS” section above can further include an amino acid substitution (such as a Y32F or Y32W substitution) at kabat position 32. In some embodiments, the Vl of any of the 10E8 variants disclosed in the “SlOOcW-DS” section above can further include an amino acid substitution (such as a Y32F or Y32W substitution) at kabat position 32.

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In some embodiments, the antibody or antigen binding fragment includes a Vh and a Vl including

SEQ ID NO: 3 (HC6-S74Y-511) and SEQ ID NO: 42 (rL3-6mut-Y32W), respectively. In some embodiments, an antibody or antigen binding fragment is provided that includes a Vh and a Vl including

SEQ ID NO: 3 (HC6-S74Y-511) and SEQ ID NO: 43 (rL3-6mut-Y32F), respectively.

In some embodiments, the antibody or antigen binding fragment includes a Vh and a Vl including SEQ ID NO: 5 (H6-51 l-4mut) and SEQ ID NO: 42 (rL3-6mut-Y32W), respectively. In some embodiments, an antibody or antigen binding fragment is provided that includes a Vh and a Vl including SEQ ID NO: 5 (H6-511-4mut) and SEQ ID NO: 43 (rL3-6mut-Y32F), respectively.

In some embodiments, the antibody or antigen binding fragment includes a Vh and a Vl including SEQ ID NO: 35 (H6-511-4mut-S100cW) and SEQ ID NO: 42 (rL3-6mut-Y32W), respectively. In some embodiments, an antibody or antigen binding fragment is provided that includes a Vh and a Vl including SEQ ID NO: 35 (H6-511-4mut-S100cW) and SEQ ID NO: 43 (rL3-6mut-Y32F), respectively.

In some embodiments, the antibody or antigen binding fragment includes a Vh and a Vl including SEQ ID NO: 38 (HC6-S74Y-511-S100cW) and SEQ ID NO: 42 (rL3-6mut-Y32W), respectively. In some embodiments, an antibody or antigen binding fragment is provided that includes a Vh and a Vl including SEQ ID NO: 38 (HC6-S74Y-511-S100cW) and SEQ ID NO: 43 (rL3-6mut-Y32F), respectively.

In some embodiments, the antibody or antigen binding fragment includes a Vh and a Vl including SEQ ID NO: 36 (H6-511-4mut-DS) and SEQ ID NO: 58 (rL3-6mut-DS-Y32W), respectively. In some embodiments, an antibody or antigen binding fragment is provided that includes a Vh and a Vl including SEQ ID NO: 36 (H6-511-4mut-DS) and SEQ ID NO: 59 (rL3-6mut-DS-Y32F), respectively.

In some embodiments, the antibody or antigen binding fragment includes a Vh and a Vl including SEQ ID NO: 39 (HC6-S74Y-511-DS) and SEQ ID NO: 58 (rL3-6mut-DS-Y32W), respectively. In some embodiments, an antibody or antigen binding fragment is provided that includes a Vh and a Vl including SEQ ID NO: 39 (HC6-S74Y-511-DS) and SEQ ID NO: 59 (rL3-6mut-DS-Y32F), respectively.

In some embodiments, the antibody or antigen binding fragment includes a Vh and a Vl including SEQ ID NO: 37 (H6-511-4mut-S100cW-DS) and SEQ ID NO: 58 (rL3-6mut-DS-Y32W), respectively. In some embodiments, an antibody or antigen binding fragment is provided that includes a Vh and a Vl including SEQ ID NO: 37 (H6-511-4mut-S100cW-DS) and SEQ ID NO: 59 (rL3-6mut-DS-Y32F), respectively.

In some embodiments, the antibody or antigen binding fragment includes a VH and a VL including SEQ ID NO: 40 (HC6-S74Y-511-S100cW-DS) and SEQ ID NO: 58 (rL3-6mut-DS-Y32W), respectively.

In some embodiments, an antibody or antigen binding fragment is provided that includes a VH and a VL including SEQ ID NO: 40 (HC6-S74Y-511-S100cW-DS) and SEQ ID NO: 59 (rL3-6mut-DS-Y32F), respectively.

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Individual heavy and light chain variable regions

Any of the following heavy chain variable regions can be “mixed and matched” with any of the 10E8 light chain (or variant thereof) variable regions disclosed herein to generate an antibody that specifically binds to gp41 and neutralizes HIV-1. Further, any of the following light chain variable regions can be “mixed and matched” with any of the 10E8 heavy chain (or variant thereof) variable regions disclosed herein to generate an antibody that specifically binds to gp41 and neutralizes HIV-1.

HC6-S74Y-511 V_H

In some embodiments, the antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs (such as SEQ ID NOs: 7, 8, and 9) and arginine, alanine, aspartate, tyrosine, lysine, threonine, threonine, and isoleucine residues at kabat positions 3, 5, 72, 74, 75, 77, 84, and 110, respectively. In additional embodiments, the antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs (such as SEQ ID NOs: 7, 8, and 9), and arginine, alanine, aspartate, tyrosine, lysine, threonine, threonine, and isoleucine residues at kabat positions 3, 5, 72, 74, 75, 77, 84, and 110, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 3 (HC6S74Y-511). In more embodiments, the antibody or antigen binding fragment comprises a Vh including the amino acid sequence set forth as SEQ ID NO: 3 (HC6-S74Y-511).

H6-511-4mut Vh

In some embodiments, the antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs (such as SEQ ID NOs: 7, 8, and 9) and arginine, glutamate, serine, lysine, aspartate, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively. In additional embodiments, the antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs (such as SEQ ID NOs:

7, 8, and 9), and arginine, glutamate, serine, lysine, aspartate, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 5 (H6-51 l-4mut). In more embodiments, the antibody or antigen binding fragment comprises a Vh including the amino acid sequence set forth as SEQ ID NO: 5 (H6-5114mut).

HC6-S74Y-511-SlOOcW V_H

In some embodiments, the antibody or antigen binding fragment comprises a Vh including the 10E8

Vh CDRs with SlOOcW substitution (such as SEQ ID NOs: 7, 8, and 62) and arginine, alanine, aspartate, tyrosine, lysine, threonine, threonine, and isoleucine residues at kabat positions 3, 5, 72, 74, 75, 77, 84, and

110, respectively. In additional embodiments, the antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs with SlOOcW substitution (such as SEQ ID NOs: 7, 8, and 62), and arginine,

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74, 75, 77, 84, and 110, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 38 (HC6-S74Y-51 l-S100cW). In more embodiments, the antibody or antigen binding fragment comprises a Vh including the amino acid sequence set forth as SEQ ID NO: 38 (HC6-S74Y-511-S100cW).

H6-511-4mut-S100cW Vh

In some embodiments, the antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs with SlOOcW substitution (such as SEQ ID NOs: 7, 8, and 62) and arginine, glutamate, serine, lysine, aspartate, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively. In additional embodiments, the antibody or antigen binding fragment comprises a Vh including the 10E8 Vh CDRs with SlOOcW substitution (such as SEQ ID NOs: 7, 8, and 62), and arginine, glutamate, serine, lysine, aspartate, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and iso leucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 35 (H6-51 l-4mut-S100cW). In more embodiments, the antibody or antigen binding fragment comprises a Vh including the amino acid sequence set forth as SEQ ID NO: 35 (H6-51 l-4mutSlOOcW).

rL3 Vl

In some embodiments, the antibody or antigen binding fragment comprises a Vl including the 10E8 Vl CDRs (such as SEQ ID NOs: 10, 11, 12) and can further include aspartate, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 7, 45, 58, 76, 83, and 85, respectively. In additional embodiments, the antibody or antigen binding fragment comprises a Vl including the 10E8 Vl CDRs (such as SEQ ID NOs: 10, 11, and 12), and aspartate, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 7, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 4 (rL3). In more embodiments, the antibody or antigen binding fragment comprises a Vl including the amino acid sequence set forth as SEQ ID NO: 4 (rL3).

rL3-6mut Vl

In some embodiments, the antibody or antigen binding fragment comprises a Vl including the 10E8

Vl CDRs (such as SEQ ID NOs: 10, 11, 12) and alanine, serine, aspartate, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 1, 2, 7, 8, 9, 16,

17, 45, 58, 76, 83, and 85, respectively. In additional embodiments, the antibody or antigen binding fragment comprises a Vl including the 10E8 Vl CDRs (such as SEQ ID NOs: 10, 11, and 12), and alanine,

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PCT/US2016/060390 serine, aspartate, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 6 (rL3-6mut). In more embodiments, the antibody or antigen binding fragment comprises a Vl including the amino acid sequence set forth as SEQ ID NO: 6 (rL3-6mut).

rL3-6mut Vl with Y32W substitution

In some embodiments, the antibody or antigen binding fragment comprises a Vl including the 10E8 Vl CDRs with Y32W substitution (such as SEQ ID NOs: 10, 11, 66) and alanine, serine, aspartate, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively. In additional embodiments, the antibody or antigen binding fragment comprises a Vl including the 10E8 Vl CDRs with Y32W substitution (such as SEQ ID NOs: 10, 11, and 66), and alanine, serine, aspartate, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 42 (rL3-6mut-Y32W). In more embodiments, the antibody or antigen binding fragment comprises a Vl including the amino acid sequence set forth as SEQ ID NO: 42 (rL3-6mut-Y32W).

rL3-6mut Vl with Y32F substitution

In some embodiments, the antibody or antigen binding fragment comprises a Vl including the 10E8 Vl CDRs with Y32F substitution (such as SEQ ID NOs: 10, 11, 67) and alanine, serine, aspartate, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively. In additional embodiments, the antibody or antigen binding fragment comprises a Vl including the 10E8 Vl CDRs with Y32F substitution (such as SEQ ID NOs: 10, 11, and 67), and alanine, serine, aspartate, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence at least 80% (such as at least 90%, or at least 95%) identical to SEQ ID NO: 43 (rL3-6mut-Y32F). In more embodiments, the antibody or antigen binding fragment comprises a Vl including the amino acid sequence set forth as SEQ ID NO: 43 (rL3-6mut-Y32F).

1. Additional description of the disclosed antibodies and antigen binding fragments

In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in CDRs. In certain embodiments of the variant Vh and Vl sequences provided herein, each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions. Amino acid substitutions (such as conservative amino acid substitutions) can be

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PCT/US2016/060390 made in the Vh and the Vl regions, for example to increase production yield or solubility. In some embodiments, the Vh of the antibody or antigen binding fragment comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared a disclosed 10E8 variant Vh, and/or the Vl of the antibody or antigen binding fragment comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared a disclosed 10E8 variant VL.

The disclosed modified 10E8 antibodies specifically bind to the MPER of gp41 at an epitope designated as the 10E8 epitope, which is included on the peptide sequence provided as NEQELLELDKWASLWNWFDITNWLWYIR (SEQ ID NO: 34). The 10E8 epitope has been elucidated by solving the structure of the 10E8 antibody in complex with a gp41 peptide (see Huang et al, Nature, 491: 406-412, 2012, incorporated by reference herein in its entirety). As described in Huang et al. (Nature, 491: 406-412, 2012) and PCT Pub. U.S. Pub. 2014/0348785A1, amino acids at Vh kabat positions 28, 31, 33, 50, 52, 52B, 52C, 53, 56, 58, and 97-100J, and Vl kabat positions 91 and 95B, of the 10E8 antibody contribute to the buried surface area of the antibody interface with gp41. Accordingly, in some embodiments, any of the modified 10E8 antibodies or antigen binding fragments disclosed herein comprises the corresponding amino acids of the parent 10E8 antibody at Vh kabat positions 28, 31, 33, 50, 52, 52B, 52C, 53, 56, 58, and 97-100J, and Vl kabat positions 91 and 95B, of the 10E8. These residues are D, N, W, R, T, P, G, E, S, D, K, Y, Y, D, F, W, S, G, Y, P, P, G, E, and E, respectively. In some embodiments, the Vh of any of the modified 10E8 antibodies or antigen binding fragments provided herein comprises an arginine residue at kabat position 50 and an aspartate residue at kabat position 58.

In some embodiments, any of the disclosed modified 10E8 antibodies or antigen binding fragments that specifically bind to gp41 and neutralize HIV-1 can further comprise one or more of the gain of function amino acid substitutions listed in any one of Tables 3, 4, 9, or 10.

In some embodiments, the antibody or antigen binding fragment can also be distinguished by neutralization breadth. For example, in some embodiments, the antibody or antigen binding fragment can neutralize at least 80% (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the HIV-1 isolates listed in FIG. 16A with an IC50 of less than 50 pg/ml. The person of ordinary skill in the art is familiar with methods of measuring the neutralization breadth and potency of an HIV-1 Env specific monoclonal antibody, for example such methods include the single-round HIV-1 Env-pseudoviruses infection of TZM-bl cells. Exemplary pseudovirus neutralization assays and panels of HIV-1 pseudovirus are described for example, in Li et al., J Virol 79, 10108-10125, 2005, Seaman et al, J. Virol., 84:1439-1452, 2010; Sarzotti-Kelsoe etal., J.

Immunol. Methods, 409:131-46, 2014; and WO2011/038290, each of which is incorporated by reference herein.

In some embodiments, the antibody or antigen binding fragment can also be distinguished by its solubility in aqueous solution. For example, in some embodiments, the antibody or antigen binding

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PCT/US2016/060390 fragment (for example, including 10E8v4 SlOOcF or 10E8v4 V5R SlOOcF variable regions) can dissolve to a concentration of at least 1.0 mg/ml (such as at least 1.0 mg/ml, at least 2.0 mg/ml, at least 3.0 mg/ml, at least 4.0 mg/ml, at least 5.0 mg/ml, at least 6.0 mg/ml, at least 7.0 mg/ml, at least 8.0 mg/ml, at least 9.0 mg/ml, or at least 10.0 mg/ml) in phosphate buffered saline (pH 7.4) at room temperature (e.g., 20-22 degrees Celsius) and remains dissolved for at least 12 hours (such as at least 24 hours, at least 48 hours, at least one week, at least two weeks, or more time). In some embodiments, the antibody or antigen binding fragment (for example, including 10E8v4 SlOOcF or 10E8v4 V5R SlOOcF variable regions) can dissolve to a concentration of at least 10 mg/ml (such as at least 15 mg/ml, at least 20 mg/ml, at least 25 mg/ml, at least 30 mg/ml, at least 35 mg/ml, at least 40 mg/ml, at least 45 mg/ml, or at least 50 mg/ml) in phosphate buffered saline (pH 7.4) at room temperature (e.g., 20-22 degrees Celsius) and remains dissolved for at least 12 hours (such as at least 24 hours, at least 48 hours, at least one week, at least two weeks, or more time). In some embodiments, the antibody or antigen binding fragment (for example, including 10E8v4 SlOOcF or 10E8v4 V5R SlOOcF variable regions) can dissolve to a concentration of at least 10 mg/ml (such as at least 15 mg/ml, at least 20 mg/ml, at least 25 mg/ml, at least 30 mg/ml, at least 35 mg/ml, at least 40 mg/ml, at least 45 mg/ml, or at least 50 mg/ml) in phosphate buffered saline (pH 7.4) at room temperature (e.g., 20-22 degrees Celsius) and remains dissolved for at least 12 hours (such as at least 24 hours, at least 48 hours, at least one week, at least two weeks, or more time). In some embodiments, the antibody or antigen binding fragment (for example, including 10E8v4 SlOOcF or 10E8v4 V5R SlOOcF variable regions) can dissolve to a concentration of at least 20 mg/ml in phosphate buffered saline (pH 7.4) at room temperature (e.g., 20-22 degrees Celsius) and remains dissolved for at least 12 hours (such as at least 24 hours, at least 48 hours, at least one week, at least two weeks, or more time). In some embodiments, the antibody or antigen binding fragment (for example, including 10E8v4 SlOOcF or 10E8v4 V5R SlOOcF variable regions) can dissolve to a concentration of at least 40 mg/ml in phosphate buffered saline (pH 7.4) at room temperature (e.g., 20-22 degrees Celsius) and remains dissolved for at least 12 hours (such as at least 24 hours, at least 48 hours, at least one week, at least two weeks, or more time). In some embodiments, the antibody or antigen binding fragment (for example, including 10E8v4 SlOOcF or 10E8v4 V5R SlOOcF variable regions) can dissolve to a concentration of at least 30 mg/ml in phosphate buffered saline (pH 7.4) at room temperature (e.g., 20-22 degrees Celsius) and remains dissolved for at least 12 hours (such as at least 24 hours, at least 48 hours, at least one week, at least two weeks, or more time). In one embodiment, the phosphate buffered saline includes NaCl (137 mM), KC1 (2.7 mM), Na₂HPO₄ (10 mM), KH2PO4 (1.8 mM) at pH 7.4. In some embodiments, the phosphate buffered saline further includes CaCl₂ (1 mM) and MgCl₂ (0.5 mM). The person of skill in the art is familiar with methods of determining if a protein remains in solution over time. For example, the concentration of the protein dissolved in an aqueous solution can be tested over time using standard methods. In some embodiments, the antibody or antigen binding fragment can also be distinguished by its auto-reactivity, that is, its binding to self antigens. For example, in some embodiments, a disclosed antibody or antigen binding fragment comprises an autoreactivity that is comparable to that of the 10E8 antibody. For example the antibody or antigen binding fragment can have not more than 10%

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PCT/US2016/060390 greater autoreactivity compared to the 10E8 antibody as measured using a standard assay, such as HEp-2 staining assay or cardiolipin binding assay.

The antibody or antigen binding fragment can be a human antibody or fragment thereof. Chimeric antibodies are also provided. The antibody or antigen binding fragment comprises any suitable framework region, such as (but not limited to) a human framework region. Human framework regions, and mutations that can be made in a human antibody framework regions, are known in the art (see, for example, in U.S. Patent No. 5,585,089, which is incorporated herein by reference). Alternatively, a heterologous framework region, such as, but not limited to a mouse or monkey framework region, can be included in the heavy or light chain of the antibodies. (See, for example, Jones et al., Nature 321:522, 1986; Riechmann et al.,

Nature 332:323, 1988; Verhoeyen et al., Science 239:1534, 1988; Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285, 1992; Sandhu, Crit. Rev. Biotech. 12:437, 1992; and Singer et al., J. Immunol. 150:2844, 1993.)

The antibody can be of any isotype. The antibody can be, for example, an IgM or an IgG antibody, such as IgGi, IgG2, IgG3, or IgG/t. The class of an antibody that specifically binds gp41 can be switched with another. In one aspect, a nucleic acid molecule encoding Vl or Vh is isolated using methods well-known in the art, such that it does not include any nucleic acid sequences encoding the constant region of the light or heavy chain, respectively. A nucleic acid molecule encoding Vl or Vh is then operatively linked to a nucleic acid sequence encoding a Cl or Ch from a different class of immunoglobulin molecule. This can be achieved using a vector or nucleic acid molecule that comprises a Cl or Ch chain, as known in the art. For example, an antibody that specifically binds gp41, that was originally IgG may be class switched to an IgM. Class switching can be used to convert one IgG subclass to another, such as from IgGi to IgG2, IgG3, or IgG/t.

In some examples, the disclosed antibodies are oligomers of antibodies, such as dimers, trimers, tetramers, pentamers, hexamers, septamers, octomers and so on.

(a) Binding affinity

In several embodiments, the antibody or antigen binding fragment can specifically bind gp41 with an affinity (e.g., measured by Kd) of no more than 1.0 χ 10⁸ M, no more than 5.0 χ 10⁸ M, no more than 1.0 χ 10⁹ M, no more than 5.0 χ 10⁹ M, no more than 1.0 χ IO¹⁰ M, no more than 5.0 χ IO¹⁰ M, or no more than 1.0 χ 10¹¹ M. Kd can be measured, for example, by a radiolabeled antigen binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen using known methods. In one assay, solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of (¹²⁵I)labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen etal., J. Mol. Biol. 293:865-881 (1999)). To establish conditions for the assay, MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 pg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 μΜ or 26 pM [ ¹²⁵Γ|antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of the anti-VEGF

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PCT/US2016/060390 antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. When the plates have dried, 150 μΐ/well of scintillant (MICROSCINT-20™; Packard) is added, and the plates are counted on a TOPCOUNT™ gamma counter (Packard) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competitive binding assays.

In another assay, Kd can be measured using surface plasmon resonance assays using a BIACORE®2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) at 25°C with immobilized antigen CM5 chips at ~10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE®, Inc.) are activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and Nhydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 pg/ml (-0.2 μΜ) before injection at a flow rate of 5 1/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25°C at a flow rate of approximately 25 l/min. Association rates (kon) and dissociation rates (koff) are calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio k_off/k_on. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 106 M^_1 s’¹ by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

(b) Multispecific antibodies

In some embodiments, the antibody or antigen binding fragment is included on a multispecific antibody, such as a bi-specific antibody or a tri-specific antibody. Such multispecific antibodies can be produced by known methods, such as crosslinking two or more antibodies, antigen binding fragments (such as scFvs) of the same type or of different types. Exemplary methods of making multispecific antibodies include those described in PCT Pub. No. WO2013/163427, which is incorporated by reference herein in its entirety. Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester) or

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PCT/US2016/060390 homobifunctional (such as disuccinimidyl suberate). Such linkers are available from Pierce Chemical

Company, Rockford, Ill.

In some embodiments, the antibody or antigen binding fragment is included on a bispecific antibody that that specifically binds to gp41 and further specifically binds to CD3. Examples of CD3 binding domains that can be included on the bispecific antibody or antigen binding fragment are known and include those disclosed in PCT Pub. No. WO2013/163427, which is incorporated by reference herein in its entirety.

Various types of multi-specific antibodies are known. Bispecific single chain antibodies can be encoded by a single nucleic acid molecule. Examples of bispecific single chain antibodies, as well as methods of constructing such antibodies are known in the art (see, e.g., U.S. Pat. Nos. 8,076,459, 8,017,748,

8,007,796, 7,919,089, 7,820,166, 7,635,472, 7,575,923, 7,435,549, 7,332,168, 7,323,440, 7,235,641,

7,229,760, 7,112,324, 6,723,538, incorporated by reference herein). Additional examples of bispecific single chain antibodies can be found in PCT application No. WO 99/54440; Mack, J. Immunol., 158:39653970, 1997; Mack, PNAS, 92:7021-7025, 1995; Kufer, Cancer Immunol. Immunother., 45:193-197, 1997; Loffler, Blood, 95:2098-2103, 2000; and Bruhl, J. Immunol., 166:2420-2426, 2001. Production of bispecific Fab-scFv (“bibody”) molecules are described, for example, in Schoonjans etal. (J. Immunol. 165:7050-57, 2000) and Willems et al. (J Chromatogr B Analyt Technol Biomed Life Sci. 786:161-76, 2003). For bibodies, a scFv molecule can be fused to one of the VL-CL (L) or VH-CH1 chains, e.g., to produce a bibody one scFv is fused to the C-term of a Fab chain.

(c) Fragments

Antigen binding fragments are encompassed by the present disclosure, such as Fab, F(ab')2, and Fv which include a heavy chain and Vl and specifically bind gp41. In several embodiments, the antigen binding fragment includes the heavy and light chain variable regions from the 10E8v4 SlOOcF or 10E8v4 V5R SlOOcF antibody.

These antibody fragments retain the ability to selectively bind with the antigen and are “antigenbinding” fragments. Non-limiting examples of such fragments include:

(1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;

(2) Fab', the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;

(3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds;

(4) Fv, a genetically engineered fragment containing the Vl and Vl expressed as two chains;

and

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PCT/US2016/060390 (5) Single chain antibody (such as scFv), defined as a genetically engineered molecule containing the Vh and the Vl linked by a suitable polypeptide linker as a genetically fused single chain molecule (see, e.g., Ahmad etal., Clin. Dev. Immunol., 2012, doi: 10.1155/2012/980250; Marbry, IDrugs, 13:543-549, 2010). The intramolecular orientation of the VH-domain and the VL-domain in a scFv, is not decisive for the provided antibodies (e.g., for the provided multispecific antibodies). Thus, scFvs with both possible arrangements (VH-domain-linker domain-VL-domain; VL-domain-linker domain-VH-domain) may be used.

(6) A dimer of a single chain antibody (scFVQ), defined as a dimer of a scFV. This has also been termed a “miniantibody.”

Methods of making these fragments are known in the art (see for example, Harlow and Lane, Antibodies: A Laboratory Manual, 2^nd, Cold Spring Harbor Laboratory, New York, 2013).

In some embodiments, the antigen binding fragment can be an Fv antibody, which is typically about 25 kDa and contain a complete antigen-binding site with three CDRs per each heavy chain and each light chain. If the Vh and the Vl are expressed non-contiguously, the chains of the Fv antibody are typically held together by noncovalent interactions. However, these chains tend to dissociate upon dilution, so methods have been developed to crosslink the chains through glutaraldehyde, intermolecular disulfides, or a peptide linker. Thus, in one example, the Fv can be a disulfide stabilized Fv (dsFv), wherein the Vh and the Vl are chemically linked by disulfide bonds. In an additional example, the Fv fragments include Vh and Vl chains connected by a peptide linker. These single-chain antigen binding proteins (scFv) can be prepared by constructing a nucleic acid molecule encoding the Vh and Vl domains connected by an oligonucleotide. The nucleic acid molecule is inserted into an expression vector, which is subsequently introduced into a host cell such as a mammalian cell. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing scFvs are known in the art (see Whitlow et al., Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991; Bird et al., Science 242:423,

1988; U.S. Patent No. 4,946,778; Pack et al., Bio/Technology 11:1271, 1993; Ahmad etal., Clin. Dev. Immunol., 2012, doi:10.1155/2012/980250; Marbry, IDrugs, 13:543-549, 2010). Dimers of a single chain antibody (scFW), are also contemplated.

Antigen binding fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in a host cell (such as an E. coli cell) of DNA encoding the fragment. Antigen binding fragments can also be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antigen binding fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab j2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly (see U.S. Patent No. 4,036,945 and U.S. Patent No. 4,331,647, and references contained therein; Nisonhoff et al., Arch. Biochem. Biophys. 89:230, 1960; Porter,

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Biochem. J. 73:119, 1959; Edelman et al., Methods in Enzymology, Vol. l,page 422, Academic Press, 1967;

and Coligan etal. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent lightheavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

Antigen binding single Vh domains, called domain antibodies (dAb), have also been identified from a library of murine Vh genes amplified from genomic DNA of immunized mice (Ward et al. Nature 341:544-546, 1989). Human single immunoglobulin variable domain polypeptides capable of binding antigen with high affinity have also been described (see, for example, PCT Publication Nos. WO 2005/035572 and WO 2003/002609). The CDRs disclosed herein can also be included in a dAb.

In some embodiments, one or more of the heavy and/or light chain complementarity determining regions (CDRs) from a disclosed antibody (such as the HC6-S74Y-511/rL3-6mut or H6-511-4mut/rL3-6mut antibody) is expressed on the surface of another protein, such as a scaffold protein. The expression of domains of antibodies on the surface of a scaffolding protein are known in the art (see e.g., Liu et al., J. Virology 85(17): 8467-8476, 2011). Such expression creates a chimeric protein that retains the binding for gp41. In some specific embodiments, one or more of the heavy chain CDRs is grafted onto a scaffold protein, such as one or more of heavy chain CDR1, CDR2, and/or CDR3. One or more CDRs can also be included in a diabody or another type of single chain antibody molecule.

(d) Variants

In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the CDRs and the framework regions. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

The variants typically retain amino acid residues necessary for correct folding and stabilizing between the Vh and the Vl regions, and will retain the charge characteristics of the residues in order to preserve the low pi and low toxicity of the molecules. Amino acid substitutions (such as conservative amino

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PCT/US2016/060390 acid substitutions) can be made in the Vh and the Vl regions, for example to increase production yield or solubility.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in CDRs. In certain embodiments of the variant Vh and Vl sequences provided above, each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.

To increase binding affinity of the antibody, the VLand Vh segments can be randomly mutated, such as within HCDR3 region or the LCDR3 region, in a process analogous to the in vivo somatic mutation process responsible for affinity maturation of antibodies during a natural immune response. Thus in vitro affinity maturation can be accomplished by amplifying Vh and Vl regions using PCR primers complementary to the HCDR3 or LCDR3, respectively. In this process, the primers have been spiked with a random mixture of the four nucleotide bases at certain positions such that the resultant PCR products encode VHand Vl segments into which random mutations have been introduced into the Vh and/or Vl CDR3 regions. These randomly mutated Vh and Vl segments can be tested to determine the binding affinity for gp4f. Methods of in vitro affinity maturation are known (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and Hoogenboom et al. inMethods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., (2001).)

A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or poly alanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex is used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

In certain embodiments, an antibody or antigen binding fragment is altered to increase or decrease the extent to which the antibody or antigen binding fragment is glycosylated. Addition or deletion of glycosylation sites may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered.

Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright etal. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose,

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N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an antibody may be made in order to create antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region; however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US

2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; W02005/053742; W02002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lee 13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533545 (1986); US Pat Appl No US 2003/0157108 Al, Presta, L; and WO 2004/056312 Al, Adams et al., especially at Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and W02003/085107).

Antibodies variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al.): U.S. Pat. No. 6,602,684 (Umana etal.): and US 2005/0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function.

Such antibody variants are described, e.g., in WO 1997/30087 (Patel etal.): WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

In several embodiments, the constant region of the antibody includes one or more amino acid substitutions to optimize in vivo half-life of the antibody. The serum half-life of IgG Abs is regulated by the neonatal Fc receptor (FcRn). Thus, in several embodiments, the antibody includes an amino acid substitution that increases binding to the FcRn. Several such substitutions are known to the person of

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PCT/US2016/060390 ordinary skill in the art, such as substitutions at IgG constant regions T250Q and M428L (see, e.g., Hinton et al., J Immunol., 176:346-356, 2006); M428L and N434S (the “LS” mutation, see, e.g., Zalevsky, etal., Nature Biotechnology, 28:157-159, 2010); N434A (see, e.g., Petkova et al., Int. Immunol., 18:1759-1769, 2006); T307A, E380A, and N434A (see, e.g., Petkova et al., Int. Immunol., 18:1759-1769, 2006); and M252Y, S254T, and T256E (see, e.g., Dall’Acqua et al., J. Biol. Chem., 281:23514-23524, 2006).The disclosed antibodies and antigen binding fragments can be linked to a Fc polypeptide including any of the substitutions listed above, for example, the Fc polypeptide comprises the M428L and N434S substitutions.

In some embodiments, the constant region of the antibody includes one of more amino acid substitutions to optimize antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC is mediated primarily through a set of closely related Fey receptors. In some embodiments, the antibody includes one or more amino acid substitutions that increase binding to FcyRIIIa. Several such substitutions are known to the person of ordinary skill in the art, such as substitutions at IgG constant regions S239D and I332E (see, e.g., Lazar etal., Proc. Natl., Acad. Sci. U.S.A., 103:4005-4010, 2006); and S239D, A330L, and I332E (see, e.g., Lazar et al., Proc. Natl., Acad. Sci. U.S.A., 103:4005-4010, 2006).

Combinations of the above substitutions are also included, to generate an IgG constant region with increased binding to FcRn and FcyRIIIa. The combinations increase antibody half-life and ADCC. For example, such combination include antibodies with the following amino acid substitution in the Fc region: (1) S239D/I332E and T250Q/M428L; (2) S239D/I332E and M428L/N434S; (3) S239D/I332E and N434A; (4) S239D/I332E and T307A/E380A/N434A; (5) S239D/I332E and M252Y / S254T/T256E; (6) S239D/A330L/I332E and 250Q/M428L; (7) S239D/A330L/I332E and M428L/N434S; (8) S239D/A330L/I332E and N434A; (9) S239D/A330L/I332E and T307A/E380A/N434A; or (10) S239D/A330L/I332E and M252Y/S254T/T256E. In some examples, the antibodies, or an antigen binding fragment thereof is modified such that it is directly cytotoxic to infected cells, or uses natural defenses such as complement, antibody dependent cellular cytotoxicity (ADCC), or phagocytosis by macrophages.

In certain embodiments, an antibody provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly1,3-dioxolane, poly-l,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules, fn general, the number and/or type of polymers used for derivatization can be determined based on considerations

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PCT/US2016/060390 including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

The antibody or antigen binding fragment can be derivatized or linked to another molecule (such as another peptide or protein). In general, the antibody or antigen binding fragment is derivatized such that the binding to gp41 is not affected adversely by the derivatization or labeling. For example, the antibody or antigen binding fragment can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (for example, a bi-specific antibody or a diabody), a detectable marker, an effector molecule, or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).

B. Conjugates

The antibodies and antigen binding fragments that specifically bind to an epitope on gp41 can be conjugated to an agent, such as an effector molecule or detectable marker, using any number of means known to those of skill in the art. Both covalent and noncovalent attachment means may be used. One of skill in the art will appreciate that various effector molecules and detectable markers can be used, including (but not limited to) toxins and radioactive agents such as ¹²⁵1, ³²P, ¹⁴C, ³H and ³⁵S and other labels, target moieties and ligands, etc. The choice of a particular effector molecule or detectable marker depends on the particular target molecule or cell, and the desired biological effect.

The choice of a particular effector molecule or detectable marker depends on the particular target molecule or cell, and the desired biological effect. Thus, for example, the effector molecule can be a cytotoxin that is used to bring about the death of a particular target cell (such as an HIV-1 infected cell). In other embodiments, the effector molecule can be a cytokine, such as IL-15; conjugates including the cytokine can be used, e.g., to stimulate immune cells locally.

The procedure for attaching an effector molecule or detectable marker to an antibody or antigen binding fragment varies according to the chemical structure of the effector. Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH2) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on a polypeptide to result in the binding of the effector molecule or detectable marker. Alternatively, the antibody or antigen binding fragment is derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any of a number of known linker molecules such as those available from Pierce Chemical Company, Rockford, IL. The linker can be any molecule used to join the antibody or antigen binding fragment to the effector molecule or detectable marker. The linker is capable of forming covalent bonds to both the antibody or antigen binding fragment and to the effector molecule or detectable marker. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibody or antigen binding fragment and the effector molecule or detectable marker are polypeptides, the linkers may

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PCT/US2016/060390 be joined to the constituent amino acids through their side groups (such as through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.

In view of the large number of methods that have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules), toxins, and other agents to antibodies one skilled in the art will be able to determine a suitable method for attaching a given agent to an antibody or antigen binding fragment or other polypeptide. For example, the antibody or antigen binding fragment can be conjugated with effector molecules such as small molecular weight drugs such as Monomethyl Auristatin E (MMAE), Monomethyl Auristatin F (MMAF), maytansine, maytansine derivatives, including the derivative of maytansine known as DM1 (also known as mertansine), or other agents to make an antibody drug conjugate (ADC). In several embodiments, conjugates of an antibody or antigen binding fragment and one or more small molecule toxins, such as a calicheamicin, maytansinoids, dolastatins, auristatins, a trichothecene, and CC1065, and the derivatives of these toxins that have toxin activity, are provided.

The antibody or antigen binding fragment can be conjugated with a detectable marker; for example, a detectable marker capable of detection by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (such as computed tomography (CT), computed axial tomography (CAT) scans, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging NMRI), magnetic resonance tomography (MTR), ultrasound, fiberoptic examination, and laparoscopic examination). Specific, nonlimiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors and the like. Bioluminescent markers are also of use, such as luciferase, Green fluorescent protein (GFP), Yellow fluorescent protein (YFP). An antibody or antigen binding fragment can also be conjugated with enzymes that are useful for detection, such as horseradish peroxidase, β- galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like. When an antibody or antigen binding fragment is conjugated with a detectable enzyme, it can be detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned. For example, when the agent horseradish peroxidase is present the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is visually detectable. An antibody or antigen binding fragment may also be conjugated with biotin, and detected through indirect measurement of avidin or streptavidin binding. It should be noted that the avidin itself can be conjugated with an enzyme or a fluorescent label.

The antibody or antigen binding fragment can be conjugated with a paramagnetic agent, such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide are also of use as labels. Antibodies can also be conjugated with lanthanides (such as europium and dysprosium), and manganese. An antibody or antigen binding fragment may also be labeled with a predetermined polypeptide epitopes recognized by a

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PCT/US2016/060390 secondary reporter (such as leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).

The antibody or antigen binding fragment can also be conjugated with a radiolabeled amino acid. The radiolabel may be used for both diagnostic and therapeutic purposes. For instance, the radiolabel may be used to detect gp41 and gp41 expressing cells by x-ray, emission spectra, or other diagnostic techniques. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ^1HIn, ¹²⁵I, ¹³¹I.

Means of detecting such detectable markers are well known to those of skill in the art. Thus, for example, radiolabels may be detected using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted illumination. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.

The average number of effector molecule or detectable marker moieties per antibody or antigen binding fragment in a conjugate can range, for example, from 1 to 20 moieties per antibody or antigen binding fragment. In certain embodiments, the average number of effector molecule or detectable marker moieties per antibody or antigen binding fragment in a conjugate range from about 1 to about 2, from about 1 to about 3, about 1 to about 8; from about 2 to about 6; from about 3 to about 5; or from about 3 to about 4. The loading (for example, effector molecule/antibody ratio) of an conjugate may be controlled in different ways, for example, by: (i) limiting the molar excess of effector molecule-linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, (iii) partial or limiting reductive conditions for cysteine thiol modification, (iv) engineering by recombinant techniques the amino acid sequence of the antibody such that the number and position of cysteine residues is modified for control of the number or position of linker-effector molecule attachments.

C. Polynucleotides and Expression

Nucleic acids molecules (for example, cDNA molecules) encoding the amino acid sequences of antibodies, antigen binding fragments, and conjugates that specifically bind gp41 are provided. Nucleic acids encoding these molecules can readily be produced by one of skill in the art, using the amino acid sequences provided herein (such as the CDR sequences and Vh and Vl sequences), sequences available in the art (such as framework or constant region sequences), and the genetic code. In several embodiments, a nucleic acid molecule can encode the Vh, the Vl, or both the Vh and Vl (for example in a bicistronic expression vector) of a disclosed antibody or antigen binding fragment. In several embodiments, the nucleic acid molecules can be expressed in a host cell (such as a mammalian cell) to produce a disclosed antibody or antigen binding fragment.

One of skill in the art can readily use the genetic code to construct a variety of functionally equivalent nucleic acids, such as nucleic acids which differ in sequence but which encode the same antibody sequence, or encode a conjugate or fusion protein including the Vl and/or Vh nucleic acid sequence.

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In a non-limiting example, an isolated nucleic acid molecule encodes the Vh of a disclosed antibody or antigen binding fragment and includes the nucleic acid sequence set forth as any one of SEQ ID NOs: 20, 21, 44, 45, 78, or 79. In a non-limiting example, an isolated nucleic acid molecule encodes the Vl of a disclosed antibody or antigen binding fragment and includes the nucleic acid sequence set forth as any one of SEQ ID NOs: 24, 46 or 47. In a non-limiting example, an isolated nucleic acid molecule encodes the Vh and Vl of a disclosed antibody or antigen binding fragment and includes the nucleic acid sequences set forth as any one of SEQ ID NOs: 44 and 46, respectively; SEQ ID NOs: 44 and 47, respectively, SEQ ID NOs: 45 and 46, respectively, SEQ ID NOs: 45 and 47, respectively (10E8v4), SEQ ID NOs: 78 and 47, respectively (10E8v4 SlOOcF), or SEQ ID NOs: 79 and 47, respectively (10E8v4 V5R, SlOOcF).

Nucleic acid sequences encoding the antibodies, antigen binding fragments, and conjugates that specifically bind gp41 can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by methods such as the phosphotriester method of Narang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109-151, 1979; the diethylphosphoramidite method of Beaucage etal., Tetra. Lett. 22:1859-1862, 1981; the solid phase phosphoramidite triester method described by Beaucage & Caruthers, Tetra. Letts. 22(20):1859-1862, 1981, for example, using an automated synthesizer as described in, for example, Needham-VanDevanter et al., Nucl. Acids Res. 12:6159-6168, 1984; and, the solid support method of U.S. Patent No. 4,458,066. Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template.

Exemplary nucleic acids can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are known (see, e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4^th ed., Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013). Product information from manufacturers of biological reagents and experimental equipment also provide useful information. Such manufacturers include the SIGMA Chemical Company (Saint Louis, MO), R&D Systems (Minneapolis, MN), Pharmacia Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (Carlsbad, CA), and Applied Biosystems (Foster City, CA), as well as many other commercial sources known to one of skill.

Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known to persons of skill.

The nucleic acid molecules can be expressed in a recombinantly engineered cell such as bacteria, plant, yeast, insect and mammalian cells. The antibodies, antigen binding fragments, and conjugates can be

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PCT/US2016/060390 expressed as individual Vh and/or Vl chain (linked to an effector molecule or detectable marker as needed), or can be expressed as a fusion protein. Methods of expressing and purifying antibodies and antigen binding fragments are known and further described herein (see, e.g., Al-Rubeai (ed), Antibody Expression and Production, Springer Press, 2011). An immunoadhesin can also be expressed. Thus, in some examples, nucleic acids encoding a Vh and Vl, and immunoadhesin are provided. The nucleic acid sequences can optionally encode a leader sequence.

To create a scFv the Vh- and VL-encoding DNA fragments can be operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3, such that the Vh and Vl sequences can be expressed as a contiguous single-chain protein, with the Vl and Vh domains joined by the flexible linker (see, e.g., Bird etal., Science 242:423-426, 1988; Huston etal., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; McCafferty etal., Nature 348:552-554, 1990; Kontermann and Dubel (Ed), Antibody Engineering, Vols. 1-2, 2^nd Ed., Springer Press, 2010; Harlow and Lane, Antibodies: A Laboratory Manual, 2^nd, Cold Spring Harbor Laboratory, New York, 2013,). Optionally, a cleavage site can be included in a linker, such as a furin cleavage site.

The nucleic acid encoding a Vh and/or the Vl optionally can encode an Fc region (immunoadhesin). The Fc region can be an IgA, IgM or IgG Fc region. The Fc region can be an optimized Fc region, as described in U.S. Published Patent Application No. 20100/093979, incorporated herein by reference. In one example, the immunoadhesin is an IgGi Fc.

The single chain antibody may be monovalent, if only a single Vh and Vl are used, bivalent, if two Vh and Vl are used, or polyvalent, if more than two Vh and Vl are used. Bispecific or polyvalent antibodies may be generated that bind specifically to gp41 and another antigen, such as, but not limited to CD3. The encoded Vh and Vl optionally comprises a furin cleavage site between the Vh and Vl domains.

Those of skill in the art are knowledgeable in the numerous expression systems available for expression of proteins including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines.

One or more DNA sequences encoding the antibodies, antigen binding fragments, or conjugates can be expressed in vitro by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art. Hybridomas expressing the antibodies of interest are also encompassed by this disclosure.

The expression of nucleic acids encoding the antibodies and antigen binding fragments described herein can be achieved by operably linking the DNA or cDNA to a promoter (which is either constitutive or inducible), followed by incorporation into an expression cassette. The promoter can be any promoter of interest, including a cytomegalovirus promoter and a human T cell lymphotrophic virus promoter (HTLV)-l. Optionally, an enhancer, such as a cytomegalovirus enhancer, is included in the construct. The cassettes can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression cassettes

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PCT/US2016/060390 contain specific sequences useful for regulation of the expression of the DNA encoding the protein. For example, the expression cassettes comprises appropriate promoters, enhancers, transcription and translation terminators, initiation sequences, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, sequences for the maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The vector can encode a selectable marker, such as a marker encoding drug resistance (for example, ampicillin or tetracycline resistance).

To obtain high level expression of a cloned gene, it is desirable to construct expression cassettes which contain, at the minimum, a strong promoter to direct transcription, a ribosome binding site for translational initiation (internal ribosomal binding sequences), and a transcription/translation terminator. For E. coli, this comprises a promoter such as the T7, trp, lac, or lambda promoters, a ribosome binding site, and preferably a transcription termination signal. For eukaryotic cells, the control sequences comprises a promoter and/or an enhancer derived from, for example, an immunoglobulin gene, HTFV, SV40 or cytomegalovirus, and a polyadenylation sequence, and can further include splice donor and/or acceptor sequences (for example, CMV and/or HTFV splice acceptor and donor sequences). The cassettes can be transferred into the chosen host cell by well-known methods such as transformation or electroporation for E. coli and calcium phosphate treatment, electroporation or lipofection for mammalian cells. Cells transformed by the cassettes can be selected by resistance to antibiotics conferred by genes contained in the cassettes, such as the amp, gpt, neo and hyg genes.

When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate coprecipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used. Eukaryotic cells can also be cotransformed with polynucleotide sequences encoding the antibody, labeled antibody, or antigen biding fragment, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Viral Expression Vectors, Springer press, Muzyczka ed., 2011). One of skill in the art can readily use an expression systems such as plasmids and vectors of use in producing proteins in cells including higher eukaryotic cells such as the COS, CHO, HeFa and myeloma cell lines.

Also provided is a population of cells comprising at least one host cell described herein. The population of cells can be a heterogeneous population comprising the host cell comprising any of the recombinant expression vectors described, in addition to at least one other cell, e.g., a host cell (e.g., a T cell), which does not comprise any of the recombinant expression vectors, or a cell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, etc. Alternatively, the population of cells can be a substantially homogeneous population, in which the population comprises mainly host cells (e.g., consisting essentially of) comprising the recombinant expression vector. The population also can be a clonal population of cells, in which all cells of the population are clones of a single host cell comprising a recombinant expression vector, such that all

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PCT/US2016/060390 cells of the population comprise the recombinant expression vector. In one embodiment of the invention, the population of cells is a clonal population comprising host cells comprising a recombinant expression vector as described herein

Modifications can be made to a nucleic acid encoding a polypeptide described herein without diminishing its biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known to those of skill in the art and include, for example, termination codons, a methionine added at the amino terminus to provide an initiation, site, additional amino acids placed on either terminus to create conveniently located restriction sites, or additional amino acids (such as poly His) to aid in purification steps. In addition to recombinant methods, the immunoconjugates, effector moieties, and antibodies of the present disclosure can also be constructed in whole or in part using standard peptide synthesis well known in the art.

Once expressed, the antibodies, antigen binding fragments, and conjugates can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, Simpson ed., Basic methods in Protein Purification and Analysis: A laboratory Manual, Cold Harbor Press, 2008). The antibodies, antigen binding fragment, and conjugates need not be 100% pure. Once purified, partially or to homogeneity as desired, if to be used therapeutically, the polypeptides should be substantially free of endotoxin.

Methods for expression of the antibodies, antigen binding fragments, and conjugates, and/or refolding to an appropriate active form, from mammalian cells, and bacteria such as E. coli have been described and are well-known and are applicable to the antibodies disclosed herein. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, 2^nd, Cold Spring Harbor Laboratory, New York, 2013, Simpson ed., Basic methods in Protein Purification and Analysis: A laboratory Manual, Cold Harbor Press, 2008, and Ward et al., Nature 341:544, 1989.

In addition to recombinant methods, the antibodies, antigen binding fragments, and/or conjugates can also be constructed in whole or in part using standard peptide synthesis. Solid phase synthesis of the polypeptides can be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid phase synthesis are described by Barany & Merrifield, The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A. pp. 3-284; Merrifield et al., J. Am. Chem. Soc. 85:2149-2156, 1963, and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, Ill., 1984. Proteins of greater length may be synthesized by condensation of the amino and carboxyl termini of shorter fragments. Methods of forming peptide bonds by activation of a carboxyl terminal end (such as by the use of the coupling reagent N, N'-dicylohexylcarbodimide) are well known in the art.

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D. Methods and Compositions

1. Therapeutic methods

Methods are disclosed herein for the prevention or treatment of an HIV-1 infection. Prevention comprises inhibition of infection with HIV-1. The methods include contacting a cell with a therapeutically effective amount of a disclosed antibody, antigen binding fragment, or conjugate that specifically binds gp41, or a nucleic acid encoding such an antibody, antigen binding fragment, or conjugate. The method can also include administering to a subject a therapeutically effective amount of a disclosed antibody, antigen binding fragment, or conjugate that specifically binds gp41, or a nucleic acid encoding such an antibody, antigen binding fragment, or conjugate, to a subject. In some examples, the antibody, antigen binding fragment, conjugate, or nucleic acid molecule can be used pre-exposure (for example, to prevent or inhibit HIV-1 infection). In some examples, the antibody, antigen binding fragment, conjugate, or nucleic acid molecule can be used in post-exposure prophylaxis. In some examples, the antibody, antigen binding fragment, conjugate, or nucleic acid molecule, can be used to eliminate or reduce the viral reservoir of HIV1 in a subject. For example a therapeutically effective amount of an antibody, antigen binding fragment, conjugate, or nucleic acid molecule can be administered to a subject with HIV-1, such as a subject being treated with anti-viral therapy. In some examples the antibody, antigen binding fragment, conjugate, or nucleic acid molecule is modified such that it is directly cytotoxic to infected cells (e.g., by conjugation to a toxin), or uses natural defenses such as complement, antibody dependent cellular cytotoxicity (ADCC), or phagocytosis by macrophages.

HIV-1 infection does not need to be completely eliminated or prevented for the method to be effective. For example, a method can decrease HIV-1 infection by a desired amount, for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination of detectable HIV-1 infected cells), as compared to HIV-1 infection in the absence of the treatment. In some embodiments, the cell is also contacted with a therapeutically effective amount of an additional agent, such as anti-viral agent. The cell can be in vivo or in vitro. The methods comprises administration of one on more additional agents known in the art. In additional embodiments, HIV-1 replication can be reduced or inhibited by similar methods. HIV-1 replication does not need to be completely eliminated for the method to be effective. For example, a method can decrease HIV-1 replication by a desired amount, for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination of detectable HIV-1), as compared to HIV-1 replication in the absence of the treatment.

Methods to assay for neutralization activity include, but are not limited to, a single-cycle infection assay as described in Martin et al. (2003) Nature Biotechnology 21:71-76. In this assay, the level of viral activity is measured via a selectable marker whose activity is reflective of the amount of viable virus in the sample, and the IC50 is determined. In other assays, acute infection can be monitored in the PM1 cell line or in primary cells (normal PBMC). In this assay, the level of viral activity can be monitored by determining the p24 concentrations using ELISA. See, for example, Martin et al. (2003) Nature Biotechnology 21:71-76.

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In one embodiment, administration of a disclosed antibody, antigen binding fragment, conjugate, or nucleic acid molecule, results in a reduction in the establishment of HIV-1 infection and/or reducing subsequent HIV-1 disease progression in a subject. A reduction in the establishment of HIV-1 infection and/or a reduction in subsequent HIV-1 disease progression encompass any statistically significant reduction in HIV-1 activity. In some embodiments, methods are disclosed for treating a subject with an HIV-1 infection. These methods include administering to the subject a therapeutically effective amount of a disclosed antibody, antigen binding fragment, conjugate, or nucleic acid molecule, thereby preventing or treating the HIV-1 infection.

Studies have shown that the rate of HIV-1 transmission from mother to infant is reduced significantly when zidovudine is administered to HIV-infected women during pregnancy and delivery and to the offspring after birth (Connor et al., 1994 Pediatr Infect Dis 714: 536-541). Several studies of mother-toinfant transmission of HIV -1 have demonstrated a correlation between the maternal virus load at delivery and risk of HIV-1 transmission to the child. The present disclosure provides antibodies, antigen binding fragments, conjugates, and nucleic acid molecule that are of use in decreasing HIV-transmission from mother to infant. Thus, in some examples, a therapeutically effective amount of a gp41-specific antibody or antigen binding fragment thereof or nucleic acid encoding such antibodies or antibody antigen binding fragments, is administered in order to prevent transmission of HIV-1, or decrease the risk of transmission of HIV-1, from a mother to an infant. In some examples, a therapeutically effective amount of the antibody, or an antigen binding fragment or nucleic acid encoding such antibody or antigen binding fragment, is administered to mother and/or to the child at childbirth. In other examples, a therapeutically effective amount of the antibody, antigen binding fragment, or nucleic acid encoding the antibody or antigen binding fragment is administered to the mother and/or infant prior to breast feeding in order to prevent viral transmission to the infant or decrease the risk of viral transmission to the infant. In some embodiments, both a therapeutically effective amount of the antibody, antigen binding fragment, or nucleic acid encoding the antibody or antigen binding fragment and a therapeutically effective amount of another agent, such as zidovudine, is administered to the mother and/or infant.

For any application, the antibody, antigen binding fragment, conjugate, or nucleic acid molecule can be combined with anti-retroviral therapy. Antiretroviral drugs are broadly classified by the phase of the retrovirus life-cycle that the drug inhibits. The disclosed antibodies can be administered in conjunction with nucleoside analog reverse-transcriptase inhibitors (such as zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, emtricitabine, entecavir, and apricitabine), nucleotide reverse transcriptase inhibitors (such as tenofovir and adefovir), non-nucleoside reverse transcriptase inhibitors (such as efavirenz, nevirapine, delavirdine, etravirine, and rilpivirine), protease inhibitors (such as saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir, fosamprenavir, atazanavir, tipranavir, and darunavir), entry or fusion inhibitors (such as maraviroc and enfuvirtide), maturation inhibitors, (such as bevirimat and vivecon), or a broad spectrum inhibitors, such as natural antivirals. In some examples, a disclosed antibody or active

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PCT/US2016/060390 fragment thereof or nucleic acids encoding such is administered in conjunction with IL-15, or conjugated to

IL-15.

Studies have shown that cocktails of HIV-1 neutralizing antibodies that target different epitopes of HIV-1 Env can treat macaques chronically infected with SHIV (Shingai et al., Nature, 503, 277-280, 2013; and Barouch et al., Nature, 503, 224-228, 2013). Accordingly, in some examples, a subject is further administered one or more additional antibodies that bind HIV-1 Env (e.g., that bind to gpl20 and/or gp41), and that can neutralize HIV-1 infection. The additional antibodies can be administrated before, during, or after administration of the novel antibodies disclosed herein (e.g., 10E8v4, 10E8v5, 10E8v4 SlOOcF, or 10E8v4 V5R SlOOcF). In some embodiments, the additional antibody can be an antibody that specifically binds to an epitope on HIV-1 Env such as the CD4 binding site (e.g., bl2, 3BNC117, VRC01, VRC07, VRC07-523 antibody), the V1/V2 domain (e.g., PG9 antibody, CAP256-VRC26 ), or the V3 loop (e.g., 101074, PGT 121, or PGT128 antibody), or those that bind both gpl20 and gp41 subunits (e.g., 35022, PGT151, or 8ANC195). In these combinatorial antibody treatment methods, the antibodies can all be in the IgG format, or another formal. Antibodies that specifically bind to these regions and neutralizing HIV-1 infection are known to the person of ordinary skill in the art. Non-limiting examples can be found, for example, in PCT Pub. No. WO 2011/038290, WO/2013/086533, WO/2013/090644, WO/2012/158948, which are each incorporated herein by reference in their entirety.

In some examples, a subject is administered the DNA encoding the antibody or antigen binding fragments thereof, to provide in vivo antibody production, for example using the cellular machinery of the subject. Immunization by nucleic acid constructs is well known in the art and taught, for example, in U.S. Patent No. 5,643,578, and U.S. Patent No. 5,593,972 and U.S. Patent No. 5,817,637. U.S. Patent No. 5,880,103 describes several methods of delivery of nucleic acids encoding to an organism. One approach to administration of nucleic acids is direct administration with plasmid DNA, such as with a mammalian expression plasmid. The nucleotide sequence encoding the disclosed antibody, or antigen binding fragments thereof, can be placed under the control of a promoter to increase expression. The methods include liposomal delivery of the nucleic acids. Such methods can be applied to the production of an antibody, or antigen binding fragments thereof, by one of ordinary skill in the art. In some embodiments, a disclosed antibody or antigen binding fragment is expressed in a subject using the pVRC8400 vector (described in Barouch et al., J. Virol, 79 ,8828-8834, 2005, which is incorporated by reference herein).

The nucleic acid molecules encoding the disclosed antibodies (such as 10E8v4 SlOOcW-DS) or antigen binding fragments can be included in a viral vector, for example for expression of the antibody or antigen binding fragment in a host cell, or a subject (such as a subject with or at risk of HIV-1 infection). A number of viral vectors have been constructed, that can be used to express the disclosed antibodies or antigen binding fragments, such as a retroviral vector, an adenoviral vector, or an adeno-associated virus (AAV) vector. In several examples, the viral vector can be replication-competent. For example, the viral vector can have a mutation in the viral genome that does not inhibit viral replication in host cells. The viral

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PCT/US2016/060390 vector also can be conditionally replication-competent. In other examples, the viral vector is replicationdeficient in host cells.

In several embodiments, a subject (such as a human subject with or at risk of HIV-f infection) can be administered a therapeutically effective amount of an adeno-associated virus (AAV) viral vector that comprises one or more nucleic acid molecules encoding a disclosed antibody or antigen binding fragment (such as 10E8v4 SlOOcF antibody). The AAV viral vector is designed for expression of the nucleic acid molecules encoding a disclosed antibody or antigen binding fragment, and administration of the therapeutically effective amount of the AAV viral vector to the subject leads to expression of a therapeutically effective amount of the antibody or antigen binding fragment in the subject. Non-limiting examples of AAV viral vectors that can be used to express a disclosed antibody or antigen binding fragment in a subject include those provided in Johnson et al (“Vector-mediated gene transfer engenders long-lived neutralizing activity and protection against SIV infection in monkeys,” Nat. Med., 15(8):901-906, 2009) and Gardner et al. (“AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges,” Nature, 519(7541): 87-91, 2015), each of which is incorporated by reference herein in its entirety.

In one embodiment, a nucleic acid encoding a disclosed antibody, or antigen binding fragments thereof, is introduced directly into cells. For example, the nucleic acid can be loaded onto gold microspheres by standard methods and introduced into the skin by a device such as Bio-Rad’s HELIOS™ Gene Gun. The nucleic acids can be “naked,” consisting of plasmids under control of a strong promoter.

Typically, the DNA is injected into muscle, although it can also be injected directly into other sites. Dosages for injection are usually around 0.5 gg/kg to about 50 mg/kg, and typically are about 0.005 mg/kg to about 5 mg/kg (see, e.g., U.S. Patent No. 5,589,466).

In another embodiment, an mRNA-based administration protocol can be used to deliver a nucleic acid encoding a disclosed antibody directly into cells. In some embodiments, nucleic acid-based therapeutics based on mRNA may provide a potent alternative to the previously mentioned approaches. mRNA therapeutics preclude safety concerns about DNA integration into the host genome and can be directly translated in the host cell cytoplasm. Moreover, the simple cell-free, in vitro synthesis of RNA avoids the manufacturing complications associated with viral vectors or cellular production and purification of complex biomolecules. Two exemplary forms of RNA-based delivery of therapeutics that can be used to deliver a nucleic acid encoding a disclosed antibody include conventional non-amplifying mRNA therapeutic delivery (see, e.g., Petsch et al., “Protective efficacy of in vitro synthesized, specific mRNA vaccines against influenza A virus infection,” Nature biotechnology, 30(12):1210-6, 2012) and selfamplifying mRNA therapeutic delivery (see, e.g., Geall et al., “Nonviral delivery of self-amplifying RNA vaccines,” PNAS, 109(36): 14604-14609, 2012; Magini et al., “Self-Amplifying mRNA Vaccines Expressing Multiple Conserved Influenza Antigens Confer Protection against Homologous and Heterosubtypic Viral Challenge,” PLoS One, 1 l(8):e0161193, 2016; and Brito et al., “Self-amplifying mRNA vaccines,” Adv Genet., 89:179-233, 2015).

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2. Dosages

A therapeutically effective amount of a gp41-specific antibody, antigen binding fragment, conjugate, or nucleic acid molecule encoding such molecules, will depend upon the severity of the disease and/or infection and the general state of the patient's health. A therapeutically effective amount is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer. The gp41-specific antibody, antigen binding fragment, conjugate, or nucleic acid molecule encoding such molecules, can be administered in conjunction with another therapeutic agent, either simultaneously or sequentially.

Single or multiple administrations of a composition including a disclosed gp41-specific antibody, antigen binding fragment, conjugate, or nucleic acid molecule encoding such molecules, can be administered depending on the dosage and frequency as required and tolerated by the patient. Compositions including the gp41-specific antibody, antigen binding fragment, conjugate, or nucleic acid molecule encoding such molecules, should provide a sufficient quantity of at least one of the gp41-specific antibody, antigen binding fragment, conjugate, or nucleic acid molecule encoding such molecules to effectively treat the patient. The dosage can be administered once, but may be applied periodically until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy. In one example, a dose of the antibody or antigen binding fragment is infused for thirty minutes every other day. In this example, about one to about ten doses can be administered, such as three or six doses can be administered every other day. In a further example, a continuous infusion is administered for about five to about ten days. The subject can be treated at regular intervals, such as monthly, until a desired therapeutic result is achieved. Generally, the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the patient.

Data obtained from cell culture assays and animal studies can be used to formulate a range of dosage for use in humans. The dosage normally lies within a range of circulating concentrations that include the ED50, with little or minimal toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The therapeutically effective dose can be determined from cell culture assays and animal studies.

In certain embodiments, the antibody or antigen binding fragment that specifically binds gp41, or conjugate thereof, or a nucleic acid molecule or vector encoding such a molecule, or a composition including such molecules, is administered at a dose in the range of from about 5 or 10 nmol/kg to about 300 nmol/kg, or from about 20 nmol/kg to about 200 nmol/kg, or at a dose of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,

55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 125, 130, 140, 150, 160, 170, 175, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 750, 1000, 1250, 1500, 1750 or 2000 nmol/kg, or at a dose of about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 pg/kg, or about 1, 1.25, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mg/kg, or other dose deemed appropriate by the treating physician. In some embodiments, the antibody or antigen binding fragment can be administered to a subject at a dose of from about 0.5 to about 40 mg/kg, such as about 1 to about 30,

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PCT/US2016/060390 about 1 to about 20, about 1 to about 15, about 1 to about 10, about 1 to about 5, about 1 to about 3, about

0.5 to about 40 mg/kg, such as about 0.5 to about 30, about 0.5 to about 20, about 0.5 to about 15, about 0.5 to about 10, about 0.5 to about 5, about 0.5 to about 3, about 3 to about 7, about 8 to about 12, about 15 to about 25, about 18 to about 22, about 28 to about 32, about 10 to about 20, about 5 to about 15, or about 20 to about 40 mg/kg. The doses described herein can be administered according to the dosing frequency/frequency of administration described herein, including without limitation daily, 2 or 3 times per week, weekly, every 2 weeks, every 3 weeks, monthly, every other month, etc.

In some embodiments, a disclosed therapeutic agent may be administered intravenously, subcutaneously, or by another mode daily or multiple times per week for a period of time, followed by a period of no treatment, then the cycle is repeated. In some embodiments, the initial period of treatment (e.g., administration of the therapeutic agent daily or multiple times per week) is for 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks. In a related embodiment, the period of no treatment lasts for 3 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. In certain embodiments, the dosing regimen of the therapeutic agent is daily for 3 days followed by 3 days off; or daily or multiple times per week for 1 week followed by 3 days or 1 week off; or daily or multiple times per week for 2 weeks followed by 1 or 2 weeks off; or daily or multiple times per week for 3 weeks followed by 1, 2, or 3 weeks off; or daily or multiple times per week for 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks followed by 1, 2, 3, or 4 weeks off.

3. Modes of Administration

The gp41-specific antibody, antigen binding fragment, conjugate, or nucleic acid molecule encoding such molecules, or a composition including such molecules, as well as additional agents, can be administered to subjects in various ways, including local and systemic administration, such as, e.g., by injection subcutaneously, intravenously, intra-arterially, intraperitoneally, intramuscularly, intradermally, or intrathecally. In an embodiment, a therapeutic agent is administered by a single subcutaneous, intravenous, intra-arterial, intraperitoneal, intramuscular, intradermal or intrathecal injection once a day. The therapeutic agent can also be administered by direct injection at or near the site of disease.

The gp41-specific antibody, antigen binding fragment, conjugate, or nucleic acid molecule encoding such molecules, or a composition including such molecules may also be administered orally in the form of microspheres, microcapsules, liposomes (uncharged or charged (e.g., cationic)), polymeric microparticles (e.g., polyamides, polylactide, polyglycolide, poly(lactide-glycolide)), microemulsions, and the like.

A further method of administration is by osmotic pump (e.g., an Alzet pump) or mini-pump (e.g., an Alzet mini-osmotic pump), which allows for controlled, continuous and/or slow-release delivery of the therapeutic agent or pharmaceutical composition over a pre-determined period. The osmotic pump or minipump can be implanted subcutaneously, or near a target site.

It will be apparent to one skilled in the art that the gp41-specific antibody, antigen binding fragment, conjugate, or nucleic acid molecule encoding such molecules, or a composition including such molecules

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PCT/US2016/060390 can also be administered by other modes. Determination of the most effective mode of administration is within the skill of the skilled artisan. The gp41-specific antibody, antigen binding fragment, conjugate, or nucleic acid molecule encoding such molecules, or a composition including such molecules can be administered as pharmaceutical formulations suitable for, e.g., oral (including buccal and sub-lingual), rectal, nasal, topical, pulmonary, vaginal or parenteral (including intramuscular, intraarterial, intrathecal, subcutaneous and intravenous) administration, or in a form suitable for administration by inhalation or insufflation. Depending on the intended mode of administration, the pharmaceutical formulations can be in the form of solid, semi-solid or liquid dosage forms, such as tablets, suppositories, pills, capsules, powders, liquids, suspensions, emulsions, creams, ointments, lotions, and the like. The formulations can be provided in unit dosage form suitable for single administration of a precise dosage. The formulations comprise an effective amount of a therapeutic agent, and one or more pharmaceutically acceptable excipients, carriers and/or diluents, and optionally one or more other biologically active agents.

4. Compositions

Compositions are provided that include one or more of the gp41-specific antibody, antigen binding fragment, conjugate, or nucleic acid molecule encoding such molecules, that are disclosed herein in a carrier. The compositions are useful, for example, for example, for the treatment or detection of an HIV-1 infection. The compositions can be prepared in unit dosage forms for administration to a subject. The amount and timing of administration are at the discretion of the treating physician to achieve the desired purposes. The gp41-specific antibody, antigen binding fragment, conjugate, or nucleic acid molecule encoding such molecules can be formulated for systemic or local administration. In one example, the gp41-specific antibody, antigen binding fragment, conjugate, or nucleic acid molecule encoding such molecules, is formulated for parenteral administration, such as intravenous administration.

In some embodiments, the compositions comprise an antibody, antigen binding fragment, or conjugate thereof, in at least 70% (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% purity. In certain embodiments, the compositions contain less than 10% (such as less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or even less) of macromolecular contaminants, such as other mammalian (e.g., human) proteins.

The compositions for administration comprises a solution of the gp41-specific antibody, antigen binding fragment, conjugate, or nucleic acid molecule encoding such molecules, dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, for example, buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the

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PCT/US2016/060390 like. The concentration of antibody in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the subject’s needs.

A typical composition for intravenous administration includes about 0.01 to about 50 mg/kg of antibody or antigen binding fragment or conjugate per subject per day (or the corresponding dose of a conjugate including the antibody or antigen binding fragment). Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, 22th ed., Pharmaceutical Press, London, UK (2012).

In some embodiments, the composition can be a liquid formulation including one or more antibodies, antigen binding fragments (such as an antibody or antigen binding fragment that specifically binds to gp41), in a concentration range from about 0.1 mg/ml to about 40 mg/ml, or from about 0.5 mg/ml to about 20 mg/ml, or from about 1 mg/ml to about 20 mg/ml, or from about 0.1 mg/ml to about 10 mg/ml, or from about 0.5 mg/ml to about 10 mg/ml, or from about 1 mg/ml to about 10 mg/ml.

Antibodies, or an antigen binding fragment thereof or a conjugate or a nucleic acid encoding such molecules, can be provided in lyophilized form and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentration. The antibody solution, or an antigen binding fragment or a nucleic acid encoding such antibodies or antigen binding fragments, can then be added to an infusion bag containing 0.9% sodium chloride, USP, and typically administered at a dosage of from 0.5 to 15 mg/kg of body weight. Considerable experience is available in the art in the administration of antibody drugs, which have been marketed in the U.S. since the approval of RlTUXAN® in 1997. Antibodies, antigen binding fragments, conjugates, or a nucleic acid encoding such molecules, can be administered by slow infusion, rather than in an intravenous push or bolus. In one example, a higher loading dose is administered, with subsequent, maintenance doses being administered at a lower level. For example, an initial loading dose of 4 mg/kg may be infused over a period of some 90 minutes, followed by weekly maintenance doses for 4-8 weeks of 2 mg/kg infused over a 30 minute period if the previous dose was well tolerated.

Controlled-release parenteral formulations can be made as implants, oily injections, or as particulate systems. For a broad overview of protein delivery systems see, Banga, A.J., Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain the therapeutic protein, such as a cytotoxin or a drug, as a central core. In microspheres the therapeutic is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 pm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 pm so that only nanoparticles are administered intravenously. Microparticles are typically around 100 pm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi,

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Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, (1992).

Polymers can be used for ion-controlled release of the antibody compositions disclosed herein. Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer, polaxamer 407, exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It has been shown to be an effective vehicle for formulation and sustained delivery of recombinant interleukin2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec etal., J. Parent. Sci. Tech. 44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215-224, 1994). In yet another aspect, liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous additional systems for controlled delivery of therapeutic proteins are known (see U.S. Patent No. 5,055,303; U.S. Patent No. 5,188,837; U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No. 4,837,028; U.S. Patent No. 4,957,735; U.S. Patent No. 5,019,369; U.S. Patent No. 5,055,303; U.S. Patent No. 5,514,670; U.S.

Patent No. 5,413,797; U.S. Patent No. 5,268,164; U.S. Patent No. 5,004,697; U.S. Patent No. 4,902,505;

U.S. Patent No. 5,506,206; U.S. Patent No. 5,271,961; U.S. Patent No. 5,254,342 and U.S. Patent No. 5,534,496).

5. Methods of detection and diagnosis

Methods are also provided for the detection of the expression of gp41 in vitro or in vivo. In one example, expression of gp41 is detected in a biological sample, and can be used to detect HIV-1 infection as the presence of HIV-1 in a sample. Uie sample can be any sample, including, but not limited to, tissue from biopsies, autopsies and pathology specimens. Biological samples also include sections of tissues, for example, frozen sections taken for histological purposes. Biological samples further include body fluids, such as blood, serum, plasma, sputum, spinal fluid or urine. Hie method of detection comprises contacting a cell or sample, or administering to a subject, an antibody or antigen binding fragment that specifically binds to gp41, or conjugate there of (e.g. a conjugate including a detectable marker) under conditions sufficient to form an immune complex, and detecting the immune complex (e.g., by detecting a detectable marker conjugated to the antibody or antigen binding fragment.

In several embodiments, a method is provided for detecting an HIV-1 infection in a subject. Hie disclosure provides a method for detecting HIV-1 in a biological sample, wherein the method comprises contacting a biological sample from a subject with a disclosed antibody or antigen binding fragment under conditions sufficient for formation of an immune complex, and detecting the immune complex, to detect the gp41 in the biological sample. In one example, the detection of gp41 in the sample indicates that the subject has an HIV-1 infection. In another example, detection of gp41 in the sample confirms a diagnosis of an HIV-1 infection in the subject.

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In some embodiments, the disclosed antibodies or antigen binding fragments thereof are used to test vaccines. For example to test if a vaccine composition including gp41 assumes a conformation including the 10E8 epitope. Thus provided herein is a method for testing a vaccine, wherein the method comprises contacting a sample containing the vaccine, such as a HIV-1 Env immunogen, with a disclosed antibody or antigen binding fragment under conditions sufficient for formation of an immune complex, and detecting the immune complex, to detect the vaccine with an HIV-1 immunogen including the 10E8 epitope in the sample. In one example, the detection of the immune complex in the sample indicates that vaccine component, such as a HIV-1 Env immunogen assumes a conformation capable of binding the antibody or antigen binding fragment.

In one embodiment, the antibody or antigen binding fragment is directly labeled with a detectable marker. In another embodiment, the antibody that binds HIV-1 Env (the first antibody) is unlabeled and a second antibody or other molecule that can bind the antibody that binds the first antibody is utilized for detection. As is well known to one of skill in the art, a second antibody is chosen that is able to specifically bind the specific species and class of the first antibody. For example, if the first antibody is a human IgG, then the secondary antibody may be an anti-human-IgG. Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially.

Suitable labels for the antibody, antigen binding fragment or secondary antibody are described above, and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. A non-limiting exemplary luminescent material is luminol; a non-limiting exemplary a magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include ¹²⁵I, ¹³¹1,³⁵S or ³H.

E. Kits

Kits are also provided. For example, kits for treating a subject with an HIV-1 infection, or for detecting gp41 in a sample or in a subject. The kits will typically include a disclosed gp41-specific antibody, antigen binding fragment, conjugate, or nucleic acid molecule encoding such molecules, or compositions including such molecules. More than one of the disclosed gp41-specific antibody, antigen binding fragment, conjugate, or nucleic acid molecule encoding such molecules, or compositions including such molecules can be included in the kit.

In one embodiment, the kit is a diagnostic kit and comprises an immunoassay. Although the details of the immunoassays may vary with the particular format employed, the method of detecting gp41 in a biological sample generally includes the steps of contacting the biological sample with an antibody which specifically reacts, under conditions sufficient to form an immune complex, to gp41. The antibody is

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PCT/US2016/060390 allowed to specifically bind under immunologically reactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.

The kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container typically holds a composition including one or more of the disclosed antibodies, antigen binding fragments, conjugates, nucleic acid molecules, or compositions. In several embodiments the container may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). A label or package insert indicates that the composition is used for treating the particular condition.

The label or package insert typically will further include instructions for use of the antibodies, antigen binding fragments, conjugates, nucleic acid molecules, or compositions included in the kit. The package insert typically includes instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products. The instructional materials may be written, in an electronic form (such as a computer diskette or compact disk) or may be visual (such as video files). The kits may also include additional components to facilitate the particular application for which the kit is designed. Thus, for example, the kit may additionally contain means of detecting a label (such as enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a secondary antibody, or the like). The kits may additionally include buffers and other reagents routinely used for the practice of a particular method. Such kits and appropriate contents are well known to those of skill in the art.

F. Additional Embodiments

Clause 1. An isolated monoclonal antibody or antigen binding fragment, comprising:

(A) a heavy chain variable region (Vh) comprising a heavy chain complementarity determining region (HCDR)l, a HCDR2, and a HCDR3 of the V_H set forth as SEQ ID NO: 35 (10E8v4 SlOOcW), and a light chain variable region (Vl) comprising a light chain complementarity determining region (LCDR)l, a LCDR2, and a LCDR3 of the V_L set forth as SEQ ID NO: 6 (rL3-6mut);

(B) a Vh comprising a HCDR1, a HCDR2, and a HCDR3 of the variable region set forth as SEQ ID NO: 36 (10E8v4 DS), and a Vl comprising a LCDR1, a LCDR2, and a LCDR3 of the variable region set forth as SEQ ID NO: 41 (rL3-6mut-DS); or (C) a Vh comprising a HCDR1, a HCDR2, and a HCDR3 of the variable region set forth as SEQ ID NO: 37 (10E8v4 SlOOcW-DS), and a V_L comprising a LCDR1, a LCDR2, and a LCDR3 of the variable region set forth as SEQ ID NO: 41 (rL3-6mut-DS); and wherein the monoclonal antibody or antigen binding fragment specifically binds to gp41 and neutralizes HIV-1.

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Clause 2. The isolated monoclonal antibody or antigen binding fragment of clause 1, wherein the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences set forth as:

SEQ ID NOs: 7, 8, 52, 10, 11, and 12 respectively (10E8v4/v5 SlOOcW IMGT CDRs);

SEQ ID NOs: 7, 8, 53, 55, 11, and 12 respectively (10E8v4/v5 DS IMGT CDRs); or

SEQ ID NOs: 7, 8, 54, 55, 5, and 6 respectively (10E8v4/v5 SlOOcW-DS IMGT CDRs).

Clause 3. The isolated monoclonal antibody or antigen binding fragment of clause 1, wherein the Vh and Vl comprise the amino acid sequences set forth as:

SEQ ID NOs: 35 and 6, respectively (10E8v4 SlOOcW);

SEQ ID NOs: 36 and 41, respectively (10E8v4 DS);

SEQ ID NOs: 37 and 41, respectively (10E8v4 SlOOcW-DS);

SEQ ID NOs: 38 and 6, respectively (10E8v5 SlOOcW);

SEQ ID NOs: 39 and 41, respectively (10E8v5 DS); or

SEQ ID NOs: 40 and 41, respectively (10E8v5 SlOOcW-DS).

Clause 4. An isolated monoclonal antibody or antigen binding fragment comprising:

(A) heavy and light chain variable regions comprising the amino acid sequences set forth as SEQ ID NOs: 5 and 6, respectively (10E8v4);

(B) heavy and light chain variable regions comprising the amino acid sequences set forth as SEQ ID NOs: 3 and 6, respectively (10E8v5);

wherein the monoclonal antibody or antigen binding fragment specifically binds to gp41 and neutralizes HIV-1.

Clause 5. The isolated antibody or antigen binding fragment of any of the prior clauses, wherein the LCDR1 further comprises an amino acid substitution of the serine residue at kabat position 32.

Clause 6. The isolated antibody or antigen binding fragment of clause 5, wherein the amino acid substitution is an Y32F substitution

Clause 7. The isolated antibody or antigen binding fragment of clause 5, wherein the amino acid substitution is an Y32W substitution.

Clause 8. The isolated monoclonal antibody or antigen binding fragment of clause 5, wherein the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences set forth as:

SEQ ID NOs: 7, 8, 9, 56, 11, and 12 respectively (10E8v4/v5 Y32W IMGT CDRs);

SEQ ID NOs: 7, 8, 52, 56, 11, and 12 respectively (10E8v4/v5 SlOOcW Y32W IMGT CDRs);

SEQ ID NOs: 7, 8, 53, 85, 11, and 12 respectively (10E8v4/v5 DS Y32W IMGT CDRs);

SEQ ID NOs: 7, 8, 54, 85, 5, and 6 respectively (10E8v4/v5 SlOOcW-DS Y32W IMGT CDRs).

SEQ ID NOs: 7, 8, 52, 57, 11, and 12 respectively (10E8v4/v5 SlOOcW Y32F IMGT CDRs);

SEQ ID NOs: 7, 8, 53, 86, 11, and 12 respectively (10E8v4/v5 DS Y32F IMGT CDRs); or

SEQ ID NOs: 7, 8, 54, 86, 5, and 6 respectively (10E8v4/v5 SlOOcW-DS Y32F IMGT CDRs).

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Clause 9. The isolated monoclonal antibody or antigen binding fragment of clause 5, wherein the Vh and Vl comprise the amino acid sequences set forth as:

SEQ ID NOs: 5 and 41, respectively (10E8v4 Y32W);

SEQ ID NOs: 35 and 6, respectively (10E8v4 SlOOcW);

SEQ ID NOs: 36 and 41, respectively (10E8v4 DS);

SEQ ID NOs: 37 and 41, respectively (10E8v4 SlOOcW-DS);

SEQ ID NOs: 3 and 41, respectively (10E8v5 Y32W);

SEQ ID NOs: 38 and 6, respectively (10E8v5 SlOOcW Y32W);

SEQ ID NOs: 39 and 41, respectively (10E8v5 DS Y32W); or

SEQ ID NOs: 40 and 41, respectively (10E8v5 SlOOcW-DS Y32W).

Clause 10. The antibody or antigen binding fragment of any of the prior clauses, further comprising one or more gain of function amino acid substitutions listed in Table 3 or Table 4.

Clause 11. The isolated antibody or antigen binding fragment of any of the prior clauses, wherein the antibody or antigen binding fragment dissolves to a concentration of at least 5 mg/ml in phosphate buffered saline, pH 7.4, at 20°DC.

Clause 12. The isolated antibody or antigen binding fragment of any of the prior clauses, wherein the antibody or antigen binding fragment neutralizes at least 95% of the viral strains listed in FIG. 16A with an IC50 of 50 pg/ml or less.

Clause 13. The antibody or antigen binding fragment of any of the prior clauses, comprising human framework regions.

Clause 14. The antibody of any of the prior clauses, comprising a human constant region.

Clause 15. The antibody of any of the prior clauses, wherein the antibody is an IgG, IgM or

IgA.

Clause 16. The antibody of any of the prior clauses, comprising a recombinant constant domain comprising a modification that increases binding to the neonatal Fc receptor.

Clause 17. The antibody of clause 16, wherein the antibody is an IgGl and the modification that increases binding to the neonatal Fc receptor comprises M428L and N434S amino acid substitutions.

Clause 18. The antigen binding fragment of any of clauses 1-13.

Clause 19. The antigen binding fragment of clause 18, wherein the antigen binding fragment is a Fv, Fab, F(ab')2, scFV or a SCFV2 fragment.

Clause 20. A bispecific antibody comprising the isolated human monoclonal antibody or antigen binding fragment of any of the preceding clauses.

Clause 21. The bispecific antibody of clause 20, wherein the antibody specifically binds to gp41 and to CD3.

Clause 22. The antibody or antigen binding fragment of any of the prior clauses, linked to an effector molecule or a detectable marker

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Clause 23. The antibody or antigen binding fragment of clause 22, wherein the detectable marker is a fluorescent, enzymatic, or radioactive marker.

Clause 24. An isolated nucleic acid molecule encoding the antibody or antigen binding fragment of any of clauses 1-21.

Clause 25. An isolated nucleic acid molecule encoding the Vh, the Vl, or the Vh and Vl, of the antibody or antigen binding fragment of any of clauses 1-21.

Clause 26. The nucleic acid molecule of any of clauses 21-24, wherein the Vh of the antibody or antigen binding fragment comprises the nucleic acid sequence set forth as SEQ ID NO: 44;

the Vh of the antibody or antigen binding fragment comprises the nucleic acid sequence set forth as SEQ ID NO: 45;

the Vl of the antibody or antigen binding fragment comprises the nucleic acid sequence set forth as SEQ ID NO: 47;

the Vh and the VLof the antibody or antigen binding fragment comprise the nucleic acid sequence set forth as SEQ ID NOs: 44 and 47, respectively; or the Vh and the VLof the antibody or antigen binding fragment comprise the nucleic acid sequence set forth as SEQ ID NOs: 45 and 47, respectively

Clause 27. The nucleic acid molecule of any of clauses 24-26, operably linked to a promoter. Clause 28. An expression vector comprising the nucleic acid molecule of any of clauses 24-27. Clause 29. The expression vector of clause 28, wherein the expression vector is a viral vector.

Clause 30. The expression vector of clause 29, wherein the viral vector is an adeno-associated viral vector.

Clause 31. A host cell comprising the nucleic acid molecule of clause 27, particularly wherein the host cell is a eukaryotic host cell such as a human host cell.

Clause 32. A pharmaceutical composition for use in treating an HIV-1 infection, comprising: a therapeutically effective amount of the antibody, antigen binding fragment, nucleic acid molecule, or expression vector of any of clauses 1-21; and a pharmaceutically acceptable carrier.

Clause 33. A method of producing an antibody or antigen binding fragment that specifically binds to gp41, comprising:

incubating the host cell of clause 31 under in vitro conditions sufficient for expression of the nucleic acid molecule or expression vector to produce the antibody or antigen binding fragment; and purifying the antibody or antigen binding fragment.

Clause 34. A method of detecting an HIV-1 infection in a subject, comprising:

contacting a biological sample from the subject with the antibody or antigen binding fragment of any of clauses 1 -23 under conditions sufficient to form an immune complex; and

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PCT/US2016/060390 detecting the presence of the immune complex on the sample, wherein the presence of the immune complex on the sample indicates that the subject has the HIV-1 infection.

Clause 35. A method of inhibiting or treating an HIV-1 infection in a subject, comprising administering to the subject a therapeutically effective amount of the antibody, antigen binding fragment, nucleic acid molecule, or expression vector of any of clauses 1-30, thereby preventing or treating the HIV-1 infection.

Clause 36. The method of clause 35, wherein the subject is at risk of an HIV-1 infection.

Clause 37. The method of clause 35, wherein the subject has an HIV- infection

Clause 38. The method of clause 37, wherein the subject has AIDS.

Clause 39. The method of any of clauses 35-38, further comprising administering to the subject an additional antibody, antigen binding fragment, or nucleic acid encoding the additional antibody or antigen binding fragment, wherein the additional antibody or antigen binding fragment specifically binds to HIV-1 Env and neutralizes HIV-1 infection.

Clause 40. The method of clause 39, wherein the additional antibody is a VRC01-class antibody.

Clause 41. The method of clause 40, wherein the VRCOl-class antibody is VRC01 or VRC07523.

Clause 42. Use of the antibody, antigen binding fragment, nucleic acid molecule, expression vector, or pharmaceutical composition of any of clauses 1-30 or 32 to inhibit or treat HIV-1 infection in a subject.

III. EXAMPLES

The following examples are provided to illustrate particular features of certain embodiments, but the scope of the claims should not be limited to those features exemplified.

Example 1: Optimized 10E8 antibodies

This example illustrates identification of modified 10E8 antibodies that have an improved combination of neutralization, solubility, and auto-reactivity properties compared to the parent 10E8 antibody.

The broadly neutralizing antibody 10E8 was isolated from the HIV-1-infected donor N152, and recognizes a helix in the membrane-proximal external region (MPER) just prior to the transmembranespanning region of the HIV-1 gp41 glycoprotein. 10E8 neutralizes 98% of diverse HIV-1 isolates with a 50% inhibitory concentration (IC50) of approximately 0.32 μ/ml, which is substantially more potent and broad than other antibodies that bind the MPER of gp41, such as the 2F5 and 4E10 antibodies. See Huang et al. (Nature, 491: 406-412, 2012) for a description of the 10E8 antibody.

Prior efforts to improve the neutralization properties of the 10E8 antibody identified the serine residue at kabat position 74 of the 10E8 Vh as a potential site of mutation (see U.S. Pub. 2014/0348785A1,

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FIG. 44). For example, a 10E8 antibody with a S74W substitution improved 10E8 neutralization 3-5 fold (see U.S. Pub. 2014/0348785A1, FIG. 45). However, when the S74W substitution was incorporated into the

HC6 Vh (discussed below) and paired with 10E8 Vl, the resulting antibody was autoreactive by cardiolipin antibody assay (see U.S. Pub. 2014/0348785A1, FIG. 56), indicating that antibodies with this substitution may not be optimal for therapeutic use.

Many clonal variants of the 10E8 heavy and light chains were identified by next generation sequencing of donor N152 samples and assessed for function by pairing with the complementary wild-type 10E8 chain (see pages 104-107 and FIGs. 50-52 of U.S. Pub. 2014/0348785A1). Among the many identified clonal variants were the HC6 Vh (referred to as gVRC-H2_dNi52 and provided as SEQ ID NO: 154 in U.S. Pub. 2014/0348785A1), the H6 V_H (referred to as gVRC-H6_dNi52 and provided as SEQ ID NO: 158 in U.S. Pub. 2014/0348785A1), the H8 V_H (referred to as gVRC-H8_dNi52 and provided as SEQ ID NO: 160 in U.S. Pub. 2014/0348785A1), the F10 V_L (referred to as gVRC-F10_dNi52 and provided as SEQ ID NO: 173 in U.S. Pub. 2014/0348785A1) and the F19 V_L (referred to as gVRC-F19dNi52 and provided as SEQ ID NO: 192 in U.S. Pub. 2014/0348785A1).

Additional modifications of the 10E8 antibody Vh were made to generate the HC6-S74Y Vh (provided as SEQ ID NO: 192 in U.S. Pub. 2014/0348785A1), and HC6-S74Y-511 V_H (referred to as HC6_S77Y_hp_F02 and provided as SEQ ID NO: 201 in U.S. Pub. 2014/0348785A1). Modifications of the 10E8 antibody Vl were made to generate the rF3 Vl (referred to as “F3,” or “10E8gF03” and provided as SEQ ID NO: 152 in U.S. Pub. 2014/0348785A1).

Despite the improvements to the 10E8 antibody described above, additional 10E8 variants that are highly soluble, lack auto-reactivity, and retain (or improve) the neutralization capability of wild-type 10E8 are needed.

The amino acid sequences of the above antibody Vh and Vl domains are provided in FIGs. 1A-1E. FIGs. 1A and IB show the CDRs of these heavy and light chain variable regions by IMGT positioning, and FIGs. 1C and ID show the CDRs of these heavy and light chain variable regions by kabat positioning. The amino acid sequences of the heavy and light chain variable regions discussed in this example are set forth in Table 2.

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Table 2. 10E8 variant sequences

V_H
10E8	SEQ ID NO	1
H6	SEQ ID NO	14
H8	SEQ ID NO	16
HC6-S74Y	SEQ ID NO	13
HC6-S74Y-511	SEQ ID NO	3
H6-511	SEQ ID NO	15
H8-511	SEQ ID NO	17
H6-511-4mut	SEQ ID NO	5
V_L
10E8	SEQ ID NO: 1
L10	SEQ ID NO: 18
L19	SEQ ID NO: 19
rL3	SEQ ID NO: 4
rL3-6mut	SEQ ID NO: 6

The modified 10E8 antibodies were produced substantially as previously described (see, e.g., Wu et al., Science, 333(6049):1593-1602, 2011 andZhu et al., Front Microbiol 3(2012):315, 2012). Briefly, antibodies were expressed by co-transfection of Expi293F cells with equal amount of the paired heavy and light chain plasmids and purified using a recombinant protein-A column (GE Healthcare). Unless context indicates otherwise, all antibody assays described in the examples utilizing variant 10E8 antibodies (having one or more amino acid substitutions in the variable regions) that were in the IgG format and were produced as discussed above.

One method for assessing antibody solubility is to examine the turbidity of a solution containing the antibody by measuring the optical density OD of the solution at 350 nm. Accordingly, the turbidity of solutions containing the 10E8 antibody, an antibody including the HC6-S74Y Vh and the rL3 Vl, an antibody including the HC6-S74Y-511 Vh and the rL3 Vl, or the VRC01 antibody (which is highly soluble in aqueous solution) was determined by measuring the OD350 of the antibody solution. Briefly, the antibodies were expressed in mammalian cells and the resulting supernatant was passed over a Protein A column. The column was washed, and bound antibody was eluted from the column using Elution Buffer (10 mM Glycine, pH 2.7) that dripped into a collection tube continuing neutralization buffer of 1M Tris-HCl, pH 8.5. As shown in FIG. 2, eluate containing the 10E8 antibody or the HC6-S74Y/rL3 antibody exhibited substantially higher OD350 readings (indicating increased turbidity) compared to the VRC01 antibody. This finding indicated that these two antibodies are prone to aggregation and less soluble than the VRC01 antibody. However, the HC6-S74Y-511/rL3 antibody was significantly less turbid (that is, more soluble) than the 10E8 and HC6-S74Y/rL3 antibodies as determined by OD350 (FIG. 2), and was nearly as effective at neutralizing HIV-1 (FIG. 3).

Additional heavy and light chain combinations were tested using the turbidity assay as a screen for solubility (FIG. 4). As shown in FIG. 4, antibodies including combinations of the H8 Vh and L10 Vl or L19

Vl, or the H6 Vh and L10 Vl all exhibited improved solubility as determined by turbidity at OD350 compared to the unmodified 10E8 antibody.

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To further improve the solubility of the H6- and H8-based antibodies, the L72,175, and F77 residues of the Vh were targeted for substitution with more hydrophilic amino acids. These residues are located within a hydrophobic patch of the 10E8 Vh (identified from the structure of 10E8/MPER complex, described in Huang etal., Nature, 491: 406-412, 2012). As shown in FIG. 4A, antibodies including the H8-511 Vh and the L10 Vl, or the H6-511 Vh and the L10 Vl exhibited greatly improved solubility compared to the unmodified 10E8 antibody, or modified 10E8 antibodies including the H6/L10, H8/L10, or H8/L19 variable regions as determined by turbidity at OD350. However, combinations of the H8-511 Vh and L10 Vl, or the H6-511 Vh and L10 Vl were each approximately 10-fold less effective for neutralization of HIV-1 viral strains (FIG. 4B). The hydrophilic substitutions were expected to reduce the overall hydrophobicity and increase solubility of the H6 and H8 Vh domains, but the dramatic reduction in neutralization potency and breadth was unexpected.

Additional modifications of the H6-511 Vh was engineered in an attempt to improve HIV-1 neutralization, without a corresponding reduction in antibody solubility. As shown in FIG. 5, residues at kabat positions 28, 31, 52, and 98 are located near the epitope binding region of 10E8. In the H6-511 Vh kabat positions 28, 31, 52, and 98 are occupied by asparagine, aspartate, serine, and histidine residues, respectively. In contrast, in the HC6 Vh (which is strongly neutralizing when paired with the rL3 Vl), kabat positions 28, 31, 52, and 98 are occupied by aspartate, asparagine, threonine, and tyrosine residues, respectively. Various combinations of kabat positions 28, 31, 52, and 98 from H6-511 Vh were substituted with the corresponding residues from the HC6 Vh to generate a series of mutant Vh domains that were paired with the rL3 Vl and tested for solubility and neutralization. As shown in FIG. 6, all the mutants exhibited improved solubility as determined by turbidity at OD350 compared to the unmodified 10E8 antibody. Further, one of the modified antibodies, H6-511-4mut (No. 7, which includes H98Y, N28D, D31N, S52T substitutions compared to H6-511) broadly neutralized HIV-1 viral strains (FIG. 7). Compared to wild-type 10E8 V_H, the H6-51 l-4mut V_H includes Q3R, A61E, P62S, E64K, L72D, S74T, I75K, F77T, L(82C)V, M84T, S87T, Y98H, R105Q, and T110I substitutions.

As shown in FIG. 8, the modified 10E8 antibodies including the L10 Vl were identified as highly soluble (compare unmodified 10E8 to 10E8H/L10). However, modified 10E8 antibodies including the L10 Vl lack the broadly neutralizing properties of the parent 10E8 antibody (FIG. 4B). In contrast, the modified 10E8 antibodies including the rL3 Vl paired with the 10E8 Vh are broad and potent neutralizers of HIV-1 infection, but are not as soluble as antibodies including the L10 Vl.

Accordingly, screening assays were performed to determine if a modified Vl could be generated with the HIV-1 neutralizing properties of the rL3 Vl, and the solubility of the L10 Vl- The L10 Vl and the rL3 Vl differ in sequence at 12 positions (see FIG. 1). Antibodies with substitutions at each of these positions were screened for solubility and neutralization properties. As shown in FIG. 9, an antibody solution containing a modified 10E8 antibody including the 10E8 Vh paired with rL3 Vl with S1A, Y2S, T8P, G9A, G16K, and R17Q substitutions was substantially less turbid than corresponding 10E8 WT or other rL3 variants. The rL3 Vl variant with SI A, Y2S, T8P, G9A, G16K, and R17Q substitutions was

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E85D substitutions compared to the native 10E8 Vl.

An antibody including the H6-51 l-4mut Vh paired with the rL3-6mut Vl was substantially more soluble (as measured using the turbidity assay) than 10E8 wt antibody or the H6-511-4mut/rL3 antibody (FIG. 10). Further, each of the H6-51 l-4mut Vh /rL3-6mut, and H6-51 l-4mut/rL3 antibodies neutralized HIV-1 with IC50 and ICso values less than that of 10E8 wt (FIG. 11).

FIG. 12 provides a summary of the development process for the HC6-S74Y-511/rL3 (Variant 1), H6-511-4mut/rL3-6mut (Variant 4), and HC6-S74Y-511/rL3-6mut (Variant5) antibodies. The nomenclature, sequence, and substitutions compared to wild-type 10E8 for these variant antibodies is summarized in Table 3.

Table 3. 10E8 variants

Antibody	Name	SEQ ID NO	Mutations compared to 10E8 Vh or Vl
Variant 1	V_H	HC6-S74Y-511	3	Q3R, V5A, L72D, S74Y, I75K, F77T, M84T, T110I
V_l	rL3	4	E7D, I45V, V58I, S76T, D83E, E85D
Variant 4	V_h	H6-511-4mut	5	Q3R, A61E, P62S, E64K, L72D, S74T, I75K, F77T, L82cV, M84T, S87T, L89Y, R105Q, T110I
V_l	rL3-6mut	6	S1A, Y2S, E7D, T8P, G9A, G16K, R17Q, I45V, V58I, S76T, D83E, E85D
Variant 5	V_h	HC6-S74Y-511	3	Q3R, V5A, L72D, S74Y, I75K, F77T, M84T, T110I
V_l	rL3-6mut	6	S1A, Y2S, E7D, T8P, G9A, G16K, R17Q, I45V, V58I, S76T, D83E, E85D

Additional assays were performed to further analyze the solubility, autoreactivity, and neutralization of the HC6-S74Y-511/rL3, H6-511-4mut/rL3-6mut, and HC6-S74Y-511/rL3-6mut antibodies (FIGs. 1316).

Kinetic concentration. A kinetic concentration assay was performed to assess solubility of the HC6-S74Y-511/rL3, H6-511-4mut/rL3-6mut, and HC6-S74Y-511/rL3-6mut antibodies. Three milliliters (cone. 0.35 OD) of each variant in phosphate buffered saline, pH 7.4 was loaded into a centrifugal concentrator with a molecule weight cut-off of 30,000 daltons, and centrifuged at 4000g for 20 minutes (FIG. 13A). After centrifugation, the volume of each of the modified antibodies was less than that of wildtype 10E8, with the volume of the H6-511-4mut/rL3-6mut and HC6-S74Y-511/rL3-6mut antibodies substantially less than that of the 10E8 antibody (FIG. 13B). A significant increase in the protein concentrations of the H6-511-4mut/rL3-6mut and HC6-S74Y-511/rL3-6mut antibody solutions was also observed (FIG. 13C).

Dynamic Light Scattering. The HC6-S74Y-511/rL3, H6-511-4mut/rL3-6mut, and HC6-S74Y51 l/rL3-6mut antibodies were assessed by dynamic light scattering. Dynamic light scattering is useful technique for determine the heterogeneity of the size of polymers dissolved in a solution. A monodisperse solution is one containing dissolved polymers (proteins) that are of equal size. A polydisperse solution contains polymers of varying size. In the case of a protein solution containing a protein that aggregates, the

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PCT/US2016/060390 dynamic light scattering assay will identify a polydisperse solution. As shown in FIG. 14, antibody solution containing wild-type 10E8 is polydisperse, whereas antibody solutions containing the HC6-S74Y-511/rF3,

H6-51 l-4mut/rF3-6mut, or HC6-S74Y-51 l/rF3-6mut antibody were monodisperse. This finding shows that these variant antibodies do not aggregate in solution.

Autoreactivity. A property common to the previously characterized MPER mAbs 2F5 and 4E10 is that they cross-react with self-antigens, and consequently are known to bind to self antigens present on HIV1 naive HEp-2 cells (Haynes etal., Science 308, 1906-1908, 2005; Huang etal., Nature, 491: 406-412, 2012). Accordingly, the auto-reactivity of the HC6-S74Y-51 l/rF3, H6-51 l-4mut/rF3-6mut, and HC6S74Y-51 l/rF3-6mut antibodies was tested by HEp-2 cell straining. Reactivity to HIV-1 negative human epithelial (HEp-2) cells was determined by indirect immunofluorescence on slides using Evans Blue as a counterstain and FITC-conjugated goat anti-human IgG (Zeus Scientific) substantially as previously described (Haynes et al., Science, 308(5730):1906-1908, 2005). As shown in FIG. 15, the HC6-S74Y511/rF3, H6-511-4mut/rF3-6mut, and HC6-S74Y-511/rF3-6mut antibodies showed little to no staining of the HEp-2 cells compared to the know autoreactive antibody VRC07-G54W. Fess staining intensity was observed for the H6-511-4mut/rF3-6mut and HC6-S74Y-511/rF3-6mut antibodies than the HC6-S74Y511/rF3 antibody.

Neutralization. To further assess the neutralizing breadth and potency of the HC6-S74Y-51 l/rF3, H6-511-4mut/rF3-6mut, and HC6-S74Y-511/rF3-6mut antibodies, these antibodies were tested on a large panel of 200 HIV-1 pseudo viruses using a previously described pseudovirus neutralization assay (see Wu et al., Science, 329(5993):856-861, 2010, incorporated by reference herein in its entirety). Briefly, monoclonal antibody was serially diluted five-fold with Dulbecco’s modified Eagle medium 10% FCS (Gibco), and 10 μΐ was incubated with 40 μΐ of pseudovirus in a 96-well plate at 37°C for 30 min. TZM-bl cells were then added and plates were incubated for 48h. Assays were then developed with a luciferase assay system (Promega), and the relative light units (RFU) were read on a luminometer (Perkin Elmer). HIV-1 Env pseudoviruses were generated by co-transfection of 293T cells with pSG3 delta Env backbone and a second plasmid that expressed HIV-1 Env at a ratio of 2:1. 72 h after transfection, supernatants containing pseudoviruses were harvested and frozen at -80°C until further use. Neutralization curves were fit by nonlinear regression using a five-parameter hill slope equation as described. The IC50 and ICso values were reported as the antibody concentrations required to inhibit infection by 50% or 80%, respectively.

Each of the 10E8, HC6-S74Y-511/rL3, H6-511-4mut/rL3-6mut, and HC6-S74Y-511/rL3-6mut antibodies neutralized 98% of the viruses in the large panel with a median IC50 of approximately 0.4 μg/ml (FIGs. 16A-16C) and a median ICsoof approximately 2.0 μg/ml, respectively (FIGs. 16D-16F).

Serum half-life. The serum half-life of the 10E8v4 antibody was assessed in macaques. Antibodies including the heavy and light chain variable regions of the 10E8 or 10E8v4 antibodies were generated with

IgGl constant region or IgGl with “FS” mutation constant region, and injected IV into macaques to assess serum half-life. As shown in FIG. 17, the 10E8v4 antibody (with or without FS substitution in the constant region) has a substantially increased half-life compared to the parent 10E8 antibody.

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In summary, the potency, breadth, and autoreactivity of the HC6-S74Y-511/rL3, H6-511-4mut/rL36mut, and HC6-S74Y-51 l/rL3-6mut antibodies are similar to that of unmodified 10E8 antibody, but the modified 10E8 antibodies are far more soluble in aqueous solution, particularly the H6-511-4mut/rL3-6mut and HC6-S74Y-51 l/rL3-6mut antibodies.

Example 2: Optimized 10E8 antibodies

This example illustrates a series of modified 10E8 antibodies developed using a surface-matrix approach where individual surface mutants were assessed for neutralization potency and manufacturability. To improve antibody 10E8, a human antibody capable of neutralizing 98% of HIV-1 isolates, 280 surface mutants were created that incorporated hydrophobic changes (Phe/Trp), hydrophilic protrusions (Arg), large steric protrusions (N-linked glycan), and side-chain removal (poly-glycine). For potency, these mutants were assessed by neutralization.

A hydrophobic substitution at heavy chain-residue 100c was observed to increase neutralization. A 10E8 variant including a SIOOcW substitution (10E8v4-100cW) yielded the broadest and most potent HIV1-neutralizing antibody yet described. Further, a poly-glycine substitution including modification of light chain-residue 32 was observed to enhance homogeneity in size exclusion chromatography, an effect also achieved by introducing a disulfide bond in this region.

1. Surface-Matrix Approach

Macromolecular function is often critically dependent on surface interactions. While alanine scanning or Arg scanning have been used to understand function, such scanning has not been used to directly improve function. Specific protein surface alterations were chosen that might enhance function.

280 surface mutants of 10E8 that incorporated hydrophobic changes (Phe/Trp - to enhance binding to hydrophobic patches), hydrophilic protrusions (Arg - to aid in solubility or to interact with membrane), large steric protrusions (N-linked glycan - to aid in solubility or to aid in steric inhibition of entry), and sidechain removal (poly-glycine - to aid in solubility or to remove steric restrictions on recognition) were generated. These 10E8 mutants were screened for HIV-1 neutralization using a 9-isolate HIV-1 panel as described above, and all except Phe/Trp changes were assessed for turbidity as described above.

Table 4. Surface Scan of 10E8 antibody

10E8 Arg Scan
Fold improvement	IC₅₀	Turbidity
Heavy chain	Light chain	Heavy chain	Light chain
> 2-fold(gain of function)	V5R	S76R
>l-fold, but <2fold (gain of function)	Q3R, S56R T68R, YlOOeR, L108R, S113R	E3R, T20R	S56R, E64R, T68R, S70R, S74R, I75R, N76R, F77R, E81R, N82aR, L89R, FlOOaR, SlOOcR, YlOOeR, P1001R, L108R, S113R	T5R, E7R, A14R, T20R, D26R, G41R, Q42R, I45R, N52R, N53R, P59R, D60R, S63R, S65R,A66R, S67R, S76R, Q79R, A80R, E81R, E85R, S94R, L95cR, G100R, T105R, L107R

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10E8 Glycan Scan
Fold improvement	ICso	Turbidity
Heavy chain	Light chain	Heavy chain	Light chain
> 2-fold (gain of function)	L170, F178, V207	S115	S21, S25, D28, S70, T75, R105, S115, S128, G134, S153, S161,L170,T191, N204	Γ8, V13, T22, S76,Q79, S94, L95c,
>l-fold, but <2fold (gain of function)	G54, S161		G54, FlOOa, L178, V207	N52, S67, Kill, S115, N129, A151, A158, K187,K205
10E8 Glycine Scan
Fold improvement	ICso & ICso	Turbidity
Heavy chain	Light chain	Heavy chain	Light chain
> 2-fold (gain of function)				(T74G, S76G and Q79G), (K103G and T105G), (T105G, L107G, S108G, Q109G, P110G andKlllG)
>l-fold, but <2fold (gain of function)	(F27F,D28G, D30G,N31G and A32G), (P52bG and E53G)		(N31G, A32G and W33G), E53G, (N82aG, N82bG, R83G, M84G and E85G), R94G, (K97g, Y98G, Y99G, D100G and FlOOaG), (D100G, FlOOaG, WIOObG, SlOOcG, YlOOeG and PlOOfG)	(Q79G, A80G, E81G and D83G),(E83G and E85G), (K93G, S92G, S95aG, R95bG and L95cG), (L95cG and V97G)
10E8 Phe/Trp Scan
Fold improvement	ICso	Turbidity
Heavy chain	Light chain	Heavy chain	Light chain
> 2-fold (gain of function)	P41F, R94F, SlOOcF
>l-fold, but <2fold (gain of function)	V5F, K13F, P52bF, F89F, FlOOaW	T5F, E7F, T8F, N52F

In the above table, “Turbidity improvement > x-fold” indicates that the actual turbidity of the variants at PBS, pH7.4 is decreased by x-fold compared to that of 10E8 antibody. For the Glycan scan, indicated residues (Ri) were substituted with Asn and the residue two position c-terminal to the indicated residue (Ri+2) was substituted with Ser or Thr to add glycan at position Ri.

A summary of mutations of interest and corresponding change in neutralization profile is provided in the following table.

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Table 5. Summary of certain 10E8 amino acid substitutions that increase HIV-1 neutralization and/or decrease turbidity.

Variant	Neutralization potency fold improvement (IC₅o/IC₈o)
HC V5R	2.8/0.6
HC S23R	2.8/0.6
LC S67R	1.9/2.3
HC S100cF	2.8/3.3
HC R94F	3.6/2.1
HC K13F	1.6/2.1
LC T5F	1.8/1.4
LC E7F	3.1/2.7
HC Glyc L178	3.4/2.7
HC Glyc L170	4.1/3.3
HC Glyc V207	3.4/1.7
HC Glyc S161	1.5/0.8
HC Glyc R105	1.2/0.96
HC Glyc S134	1.02/0.74
HC Glyc S153	1.1/0.7

2. Substitution at light chain kabat position 32

When 10E8, 10E8 variant 4 and 10E8 variant 5 are purified using gel chromatography, an unusual “double peak” is observed (see FIG. 18A). Although this double peak does not appear to affect neutralization breadth or potency, or antibody solubility, additional modifications of 10E8 antibody sequence were pursued to determine if the double peak could be removed. Based on the structure of 10E8 bound to MPER peptide, many amino acid substitutions were assayed, including kabat position 32 of the Vl.

Surprisingly, substitution of the tyrosine residue at kabat position 32 of the Vl with nearly any other amino acid removed the “double peak” (FIGs. 18B and 18C). The resulting 10E8 variants were assayed for HIV-1 neutralization using the 9-isolate HIV-1 panel, specifically chosen to represent diversity. As indicated in the tables below, a 10E8 variant 4 antibody further including a Y32W substitution has improved HIV-1 neutralization properties compared to 10E8 variant 4, and a variant with a Y32F substitution was nearly as potent as 10E8 variant 4.

Table 6. Neutralization profile of 10E8 variants

clad e	Virus	Antibody
10E8v4 V_L Y32D	10E8v4 V_L Y32E	10E8v4 V_L Y32H	10E8v4 V_L Y32K	10E8v4 V_L Y32N	10E8v4 V_L Y32Q	10E8v4 V_L Y32R	10E8v4 V_L Y32T	10E8 v4	10E8 wt
A	KER2008.12.S G3	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
AC D	6095.V1.C10.S G3	1.550	0.215	0.032	0.359	2.920	0.281	0.152	0.145	0.001	0.001
AE	TH966.8.SG3	>50	19.000	0.788	10.200	>50	11.500	4.080	3.760	0.021	0.023
B	6101.10.SG3	>50	>50	7.400	>50	>50	>50	>50	>50	0.007	0.001
B	PVO.04.SG3	>50	>50	>50	>50	>50	>50	>50	>50	1.130	0.997
B	YU2.DG.SG3	>50	>50	8.760	>50	>50	>50	>50	43.500	0.386	0.280

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c	CNE31.SG3	>50	>50	29.900	>50	>50	>50	>50	>50	1.160	0.995
c	ZM106.9.SG3	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
c	ZM215.8.SG3	>50	>50	1.390	33.000	>50	>50	7.520	9.250	0.013	0.018

Median IC50	1.550	9.608	4.395	10.200	2.920	5.891	4.080	6.505	0.021	0.023
Geometric Mean	1.550	2.021	2.020	4.944	2.920	1.798	1.671	3.849	0.048	0.033
clad e	Virus	Antibody
10E8v4 V_L Y32A	10E8v4 V_L Y32F	10E8v4 V_L Y32I	10E8v4 V_L Y32L	10E8v4 V_L Y32M	10E8v4 V_L Y32P	10E8v4 V_L Y32V	10E8v4 V_L Y32W	10E8 v4	10E8 wt
A	KER2008.12.S G3	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
AC D	6095.V1.C10.S G3	0.409	0.017	1.300	0.593	1.030	3.200	0.558	0.005	0.014	0.012
AE	TH966.8.SG3	3.530	0.031	21.400	4.260	29.800	>50	4.960	0.029	0.036	0.032
B	6101.10.SG3	>50	0.080	>50	38.600	>50	>50	>50	0.055	0.042	0.014
B	PVO.04.SG3	>50	2.250	>50	>50	>50	>50	>50	1.400	2.390	2.090
B	YU2.DG.SG3	>50	2.000	>50	>50	>50	>50	>50	1.070	1.600	1.050
C	CNE31.SG3	>50	3.200	>50	>50	>50	>50	>50	0.719	1.160	1.190
C	ZM106.9.SG3	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
C	ZM215.8.SG3	11.700	0.102	>50	8.750	>50	>50	28.200	0.089	0.070	0.085

Median IC50	3.53	0.102	11.35	6.505	15.415	3.2	4.96	0.089	0.07	0.085
Geometric Mean	2.56583	0.25052	5.2744 7	5.40462	5.54022	3.2	4.27355	0.13372	0.1818 4	0.142 5

Several of the 10E8 variant 4 antibodies that further include a substitution at Vl kabat position 32 were also assayed for turbidity (FIG. 18D). 10E8 variant 4 with Y32A, Y32F, Y32I, Y32L, Y32M, Y32P, Y32V, or Y32W substitution each had a turbidity profile similar to that of 10E8 variant 4.

2.10E8v4-100cW

The surface-matrix mutagenesis assay discussed above identified a hydrophobic change at kabat position 100c of the heavy chain to improve HIV-1 neutralization potency (the SlOOcF substitution). Analysis of the antibody surface suggested potential membrane interaction, and that a Trp substitution at kabat position 100c might also be of interest. Accordingly, a SlOOcW substitution was introduced into

10E8v4 and analyzed for HIV-1 neutralization (FIG. 19). Testing 10E8v4-100cW on a panel of 208 HIV-1 isolates indicated it to be the most potent and broadest antibody yet identified.

ANA Hep-2 staining analysis and anti-cardiolipin ELISA were performed to test the auto-reactivity of several 10E8 variants. The Hep-2 staining assays were performed as described above. The anti15 cardiolipin ELISA was performed as described in Asokan et al. J. Virol., doi: 10.1128/JVI.02097-15, 2015). The results of these assays are summarized in the following table.

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Table 7. Autoreactivity of 10E8 variants

Antibody	ANA HEp2 staining assay	Anti-cardiolipin ELISA
10E8 wt	Non-reactive	Non-reactive
10E8v4	Non-reactive	Non-reactive
10E8v4 w/ LS heavy chain	Non-reactive	Non-reactive
10E8v4 slOOcW	Non-reactive	Non-reactive
10E8v4 Y32W	Non-reactive	Non-reactive
10E8v4 Y32F	Non-reactive	Non-reactive
10E8v4 S100cW/Y32F	Non-reactive	Non-reactive
10E8v4 S100cW/Y32W	Reactive (1+)	Non-reactive
10E8v4 SlOOcF	Non-reactive	Non-reactive
10E8v4 S100cF/Y32F	Non-reactive	Non-reactive
10E8v4 S100cF/Y32W	Non-reactive	Non-reactive
10E8v4 SlOOcY	Non-reactive	Non-reactive
10E8v4 S100cY/Y32F	Non-reactive	Non-reactive
10E8v4 S100cY/Y32W	Non-reactive	Non-reactive

3.10E8v4-100cW-DS

An atypical purification profile for 10E8v4 lOOcW was observed by size exclusion chromatography. Accordingly, the purification profile of several additional 10E8 variants was assayed by size exclusion chromatography. The variants included:

1. 10E8v4 SlOOcF

2. 10E8v4 SlOOcF with an A, D, E, F, Η, I, K, L, Μ, N, P, Q, R, Τ, V, or W substitution at Vl kabat position 32

3. 10E8v4 SlOOcW

4. 10E8v4 SlOOcW with an A, D, E, F, Η, I, K, L, Μ, N, P, Q, R, Τ, V, or W substitution at Vl kabat position 32

5. 10E8v4 with a D, E, I, K, Μ, N, P, Q, T, or V substitution at Vh kabat position 100c

6. 10E8v4-DS (V_H YlOOeC and V_L S30C substitutions)

7. 10E8v4 SlOOcW-DS

8. 10E8v4-HCDR3 (HCDR3 deletion)

Of these modified 10E8 antibodies, 10E8v4 SlOOcP and 10E8v4 HCDR3 showed a typical antibody profile based on size exclusion chromatography, and 10E8v4 S100cF/Y32N, 10E8v4 S100cW/Y32N, 10E8v4-DS, and 10E8v4 SlOOcW-DS showed a nearly typical antibody profile based on size exclusion chromatography. The remaining antibodies eluted from the size exclusion column in multiple peaks. A summary of the size exclusion chromatography profiles for the 10E8v4, 10E8v4/Y32F, 10E8v4 SlOOcA, 10E8v4 with HCDR3 deletion, 10E8v4-DS, and 10E8v4 SlOOcW-DS antibodies is provided in FIGs. 20 and 21. A brief summary of the size exclusion chromatography assays is provided below:

System: Acquity Waters UPLC H-class;

Column: Acquity UPLC Protein BEH200 SEC, Particle Size: 200A, Pore Size: 1.7pm, Column Dimension: 4.6mm x 150mm

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UV Absorbance: 280nm

Mobile Phase: 2X PBS

Flow Rate: 0.4mL/min (isocratic)

Run Time: 20 minutes

Sample Volume Injected: 50u L

Sample Concentration: lmg/mL Sample Mass: 50pg The Sample was ran in duplicate.

Control 1: VRC01 D-13-0030A, lmg/mL; Volume injected: 50liL

Control 2: Gel Filtration Standard, 9mg/mL; Volume injected: 30liL

The ability of the following variants to bind to the MPER peptide was assessed: 10E8v4

S100cF/LC-Y32N, 10E8v4 S100cW/LC-Y32N, 10E8v4 SlOOcP, 10E8v4-DS, and 10E8v4 HCDR3 (10E8v4 w/HCDR3 deletion). The binding of all was severely impaired, except for 10E8v4_DS, which bound 2-fold more tightly than 10E8v4. 10E8v4-100cW-DS binding to MPER peptide was also assessed and found to be similar to that of 10E8v4-DS.

Several of these 10E8 variants were assayed for HIV-1 neutralization using the 9-isolate HIV-1 panel, specifically chosen to represent diversity, with the results shown in the table below.

Table 8. HIV-1 neutralization of 10E8 variants.

clade	Virus	IC₅₀	ICeo
10E8v4	10E8v4 DS	10E8v4 lOOcW	10E8v4 lOOcW DS	10E8v4	10E8v4 DS	10E8v4 lOOcW	10E8v4 lOOcW DS
A	KER2008.12.SG 3	>50	>50	>50	>50	>50	>50	>50	>50
ACD	6095.V1.C10.SG 3	0.004	0.002	0.000	0.002	0.021	0.017	0.001	0.013
AE	TH966.8.SG3	0.026	0.092	0.003	0.019	0.169	0.345	0.013	0.092
B	6101.10.SG3	0.011	2.410	0.002	1.220	0.090	22.200	0.010	10.100
B	PVO.04.SG3	0.894	2.530	0.085	0.331	8.000	10.100	0.459	3.670
B	YU2.DG.SG3	0.668	0.275	0.031	0.213	5.320	2.440	0.260	2.200
C	CNE31.SG3	1.740	2.280	0.107	0.806	4.850	6.480	0.295	2.930
C	ZM106.9.SG3	>50	>50	>50	>50	>50	>50	>50	>50
C	ZM215.8.SG3	0.042	0.208	0.004	0.037	0.326	0.798	0.025	0.245

Median IC50 or IC80	0.042	0.275	0.004	0.213	0.326	2.440	0.025	2.200
Geometric Mean	0.091	0.283	0.008	0.100	0.578	1.494	0.038	0.684

Example 3: Detecting HIV-1 in a sample or a subject using a gp41-specific antibody

This example describes the use of HIV-1 monoclonal neutralizing antibodies specific to gp41 for the detection of HIV-1 in a sample or a subject. This example further describes the use of these antibodies to confirm the diagnosis of HIV-1 infection in a subject.

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A biological sample, such as a blood sample, is obtained from the patient diagnosed with, undergoing screening for, or suspected of having an HIV-1 infection. A blood sample taken from a patient who is not infected is used as a control, although a standard result can also be used as a control. An ELISA is performed to detect the presence of gp41 in the blood sample. Proteins present in the blood samples (the patient sample and control sample) are immobilized on a solid support, such as a 96-well plate, according to methods well known in the art (see, for example, Robinson et al., Lancet 362:1612-1616, 2003, incorporated herein by reference). Following immobilization, HIV-1 monoclonal neutralizing antibodies specific to gp41 that are directly labeled with a fluorescent marker are applied to the protein-immobilized plate. The plate is washed in an appropriate buffer, such as PBS, to remove any unbound antibody and to minimize nonspecific binding of antibody. Fluorescence can be detected using a fluorometric plate reader according to standard methods. An increase in fluorescence intensity of the patient sample, relative to the control sample, indicates the gp41 antibody specifically bound proteins from the blood sample, thus detecting the presence of gp41 protein in the sample. Detection of gp41 protein in the patient sample indicates the patient has an HIV-1 infection, or confirms diagnosis of HIV-1 infection in the subject.

Example 4: Treatment of HIV-1 using a monoclonal antibody specific for gp41

This example describes a particular method that can be used to treat HIV-1 infection in a human subject by administration of one or more gp41-specific human neutralizing antibodies. Although particular methods, dosages, and modes of administrations are provided, one skilled in the art will appreciate that variations can be made without substantially affecting the treatment.

Based upon the teaching disclosed herein, HIV-1 infection can be treated by administering a therapeutically effective amount of one or more of the neutralizing antibodies described herein, thereby reducing or eliminating HIV-1 infection.

Screening subjects: In particular examples, the subject is first screened to determine if they have an HIV-1 infection. Examples of methods that can be used to screen for HIV-1 infection include a combination of measuring a subject’s CD4+ T cell count and the level of HIV-1 virus in serum blood levels. Additional methods using the gp41-specific antibodies described herein can also be used to screen for HIV-1 infection.

In some examples, HIV-1 testing consists of initial screening with an enzyme-linked immunosorbent assay (ELISA) to detect antibodies to HIV-1. Specimens with a nonreactive result from the initial ELISA are considered HIV-1-negative unless new exposure to an infected partner or partner of unknown HIV-1 status has occurred. Specimens with a reactive ELISA result are retested in duplicate. If the result of either duplicate test is reactive, the specimen is reported as repeatedly reactive and undergoes confirmatory testing with a more specific supplemental test (e.g., Western blot or an immunofluorescence assay (IFA)). Specimens that are repeatedly reactive by ELISA and positive by IFA or reactive by Western blot are considered HIV-positive and indicative of HIV-1 infection. Specimens that are repeatedly ELISA-reactive occasionally provide an indeterminate Western blot result, which may be either an incomplete antibody response to HIV-1 in an infected person, or nonspecific reactions in an uninfected person. IFA can be used

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The detection of HIV-1 in a subject’s blood is indicative that the subject is infected with HIV-1 and is a candidate for receiving the therapeutic compositions disclosed herein. Moreover, detection of a CD4+ T cell count below 350 per microliter, such as 200 cells per microliter, is also indicative that the subject is likely to have an HIV-1 infection.

Pre-screening is not required prior to administration of the therapeutic compositions disclosed herein

Pre-treatment of subjects: In particular examples, the subject is treated prior to administration of a therapeutic agent that includes one or more antiretroviral therapies known to those of skill in the art. However, such pre-treatment is not always required, and can be determined by a skilled clinician.

Administration of therapeutic compositions: Following subject selection, a therapeutically effective dose of a gp41-specific neutralizing antibody described herein (such as the H6-51 l-4mut/rL3-6mut antibody) is administered to the subject (such as an adult human or a newborn infant either at risk for contracting HIV-1 or known to be infected with HIV-1). Additional agents, such as anti-viral agents, can also be administered to the subject simultaneously or prior to or following administration of the disclosed agents. Administration can be achieved by any method known in the art, such as oral administration, inhalation, intravenous, intramuscular, intraperitoneal, or subcutaneous.

The amount of the composition administered to prevent, reduce, inhibit, and/or treat HIV-1 or a condition associated with it depends on the subject being treated, the severity of the disorder, and the manner of administration of the therapeutic composition. Ideally, a therapeutically effective amount of an agent is the amount sufficient to prevent, reduce, and/or inhibit, and/or treat the condition (e.g., HIV-1) in a subject without causing a substantial cytotoxic effect in the subject. An effective amount can be readily determined by one skilled in the art, for example using routine trials establishing dose response curves. As such, these compositions may be formulated with an inert diluent or with a pharmaceutically acceptable carrier.

In one specific example, antibodies are administered at 5 mg per kg every two weeks or 10 mg per kg every two weeks depending upon the particular stage of HIV-1. In an example, the antibodies are administered continuously. In another example, antibodies or antibody fragments are administered at 50 pg per kg given twice a week for 2 to 3 weeks.

Administration of the therapeutic compositions can be taken long term (for example over a period of months or years).

Assessment: Following the administration of one or more therapies, subjects with HIV-1 can be monitored for reductions in HIV-1 levels, increases in a subject’s CD4+ T cell count, or reductions in one or more clinical symptoms associated with HIV-1. In particular examples, subjects are analyzed one or more times, starting 7 days following treatment. Subjects can be monitored using any method known in the art.

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For example, biological samples from the subject, including blood, can be obtained and alterations in HIV-1 or CD4+ T cell levels evaluated.

Additional treatments: In particular examples, if subjects are stable or have a minor, mixed or partial response to treatment, they can be re-treated after re-evaluation with the same schedule and preparation of agents that they previously received for the desired amount of time, including the duration of a subject’s lifetime. A partial response is a reduction, such as at least a 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 70% in HIV-1 infection, HIV-1 replication or combination thereof. A partial response may also be an increase in CD4+ T cell count such as at least 350 T cells per microliter.

Example 5

Optimized 10E8 antibodies

This example describes arginine and tryptophan scanning mutagenesis assays to identity variants of the 10E8 V5R SlOOcF antibody (which include a VH and VL set forth as SEQ ID NOs: 76 and 6) with improved HIV-1 neutralization.

Heavy chain variable region residues El, V5, S7, G8, G10, LI 1, K13, G15, G16, S17, S21, S23,

S25, D28, D30, N31, P41, G42, K43, E46, P52b, G52c, E53, G54, W55, S56, E61, S62, K64, G65, T68,

S70, D72, T74, K75, N76, E81, N82a, N82b, T84, E85, Y89, K97, Y99, FlOOa, WIOOb, YlOOe, PlOOf, PlOOg, Q101, D102, Q105, L108, and SI 13 (kabat numbering) of the 10E8v4 V5R SlOOcF antibody were individually mutated to arginine or tryptophan. Additionally, light chain variable region residues S2, E3, T5, D7, P8, A9, S12, A14, L15, K16, Q17, T18, T20, D26, S30, P40, G41, Q42, V45, K51, N52, S56, G57, P59, D60, S63, S65, A66, S67, G68, N69, T76, G77, Q79, A80, E81, D85, K93, S94, G100, K103, and L107 (kabat numbering) of the 10E8v4 V5R SlOOcF antibody were individually mutated to arginine or tryptophan. Thus, a total of 172 10E8v4 V5R SlOOcF mutants were generated. Each of the resulting 10E8v4 V5R SlOOcF mutants was expressed (as an IgG) and tested using the pseudovirus neutralization assay described above. Of these, the following variants were identified to improve HIV-1 neutralization (by geometric mean) at least 2-fold compared to the parent 10E8v4 V5R SlOOcF antibody:

Table 9. 10E8 variants.

Fold improvement	Geometric mean IC50	Geometric mean ICso
Vh substitution	Vl substitution	Vh substitution	Vl substitution
> 4-fold, but <5-fold				G68R
>3-fold, but <4-fold	E1R, D28W, P52bR, D72R	D7W, D26R
> 2-fold, but < 3-fold	G15R, D30R, N31R, E81R, E1W, S25W, D30W, N31W	A14R, T18R		Q17R, T18R, T20R, K51R, N53R, S65R, G77R, G100R

The 10E8v4 V5R SlOOcF variants that led to at least a 3 fold improvement in HIV-1 neutralization were positioned within the Cryo-EM HIV-1 Env structure fitted with the 10E8v4 antibody (see FIG. 22).

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This analysis revealed that some of the identified mutations clustered together on the 10E8 antibody. One group of mutations was in a membrane-proximal region of 10E8 when 10E8 is bound to HIV-1 Env (D7W,

D26R, and G68R substitutions to the Vl), and another group was in a region of 10E8 that is not proximal to the cell membrane (D72R, E1R D28W, and P52bR substitutions to the Vh). These groups of substitutions can be introduced into the sequence of the 10E8v4 V5R SlOOcF antibody to generate additional 10E8 variants as follows:

10E8v4 V5R SlOOcF with D72R, E1R D28W, and P52bR substitutions to the Vh;

10E8v4 V5R SlOOcF with D7W, D26R, and G68R substitutions to the Vl;

10E8v4 V5R SlOOcF with D72R, E1R D28W, and P52bR substitutions to the V_HandD7W, D26R, 10 and G68R substitutions to the Vl;

10E8v4 V5R SlOOcF variants that include combinations of the mutations identified in the individual mutation screens were then generated and assayed for HIV-1 neutralization. Of the combination of interest identified (see the following table), several were expressed and tested using the pseudovirus neutralization assay described above. Of these, several variants were identified to improve HIV-1 neutralization compared to the parent 10E8v4 V5R SlOOcF antibody (see FIGs. 23A-23F).

Table 10. 10E8 variants.

Name of combination	Substitutions in VH compared to 10E8v4 V5R SlOOcF VH (SEQ ID NO: 76)	Substitutions in VL compared to 10E8v4 VL (SEQ ID NO: 6)
10E8-1053	E1R	D7W
10E8-1054	E1R	D26R
10E8-1055	E1R	G68R
10E8-1056	D28W	D7W
10E8-1057	D28W	D26R
10E8-1058	D28W	G68R
	D28W	Q17R
	D28W	T18R
	D28W	T20R
	D28W	K51R
	D28W	G77R

10E8-1059	P52bR	D7W
10E8-1060	P52bR	D26R
10E8-1061	P52bR	G68R
	P52bR	Q17R
	P52bR	T18R
	P52bR	T20R
	P52bR	K51R
	P52bR	G77R
10E8-1062	D72R	D7W
10E8-1063	D72R	D26R
10E8-1064	D72R	G68R

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10E8-1065	D28F	D7W
10E8-1066	D28F	D26R
10E8-1067	D28F	G68R
10E8-1068	D28F	D7W + D26R
10E8-1069	D28F	D7W + G68R
10E8-1070	D28F	D26R + G68R
10E8-1071	D28F	D7W + D26R + G68R
10E8-1072	D28F	No change
10E8-1073	D28Y	No change
10E8-1074	D30F
10E8-1075	D30Y
10E8-1076	N31F
10E8-1077	N31Y
10E8-1078	E1F
10E8-1079	E1Y
10E8-1080	P52bR	D7W + D26R
10E8-1081	P52bR	D7W + G68R
10E8-1082	P52bR	D26R + G68R
10E8-1083	P52bR	D7W + D26R + G68R
10E8-1084	P52bR	No change
10E8-1085	P52bR
10E8-1086	D28F	Q17R
10E8-1087	D28F	T18R
10E8-1088	D28F	T20R
10E8-1089	D28F	K51R
10E8-1090	D28F	G77R
10E8-1091	P52bR	Q17R
10E8-1092	P52bR	T18R
10E8-1093	P52bR	T20R
10E8-1094	P52bR	K51R
10E8-1095	P52bR	G77R

10E8-1022 (10E8v4 V5R SlOOcF)	No change	No change

Several variants listed had an improved neutralization profile compared to the patent 10E8v4V5R SlOOcF antibody, including 10E8-1081, which was about two fold more potent compared to 10e8v4 V5R lOOcF (10E8-1022), and 10E8-1073, which neutralized all the viruses in the panel with an IC50 value of less than 25 pg/ml.

Example 6

Antibody Characterization

Assays were performed with the 10E8v4, 10E8v4 SlOOcW, 10E8v4 SlOOcF, and 10E8v4 V5R 10 SlOOcF antibodies to determine the appropriateness of each of these antibodies for clinical production. The

10E8 variants were produced by transient expression in CHO cells, purified using standard techniques, and subjected to a panel of assays concerning the physical characteristics of the purified antibody. The expressed antibodies included the 10E8v4, 10E8v4 SlOOcW, 10E8v4 SlOOcF, and 10E8v4 V5R SlOOcF V_H and VL

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1. Size Exclusion Chromatography (SEC) to determine the level of aggregation and multimerization of the purified 10E8 variants. Each of the 10E8 variants was primarily in one peak coming off the size exclusion column.

2. Size Exclusion Chromatography (SEC) following 5 freeze thaw cycles to determine if freeze stress would induce antibody aggregation. The freeze thaw cycles did not significantly alter the elution profile of the 10E8 variants from the size exclusion column.

3. The appearance of the antibody solution was evaluated following initial purification. All antibody solutions were clear, colorless, and had no visible particulate.

4. Antibody Concentration (UV-VIS): The concentration of the antibody in solution following initial purification determined by absorbance (A) at 280 nm (characteristic of proteins) and light scattering (OD) due to sub-visible/visible particles in the solution at 350 nm. Low OD350 nm (<0.05 OD) values indicated minimal large particles in the starting material.

5. Thermal Transition measured by Differential Scanning Calorimetry (DSC): These measurements determined that the structural transition temperatures of the 10E8 variants fell within a normal range.

6. Circular Dichroism (CD): CD assays were performed to determine if the secondary structure of the 10E8 variants falls within a normal range. As shown in the table, the CD assays identified CD signal minimums at about 218 nm and 230 nm. Overall the CD results indicate that the 10E8 variants have somewhat typical secondary structure for an antibody. Similar CD measurements were obtained for each variant.

7. Dynamic Light Scattering (DLS): DLS was used to evaluate heterogeneity of population size for the variant antibodies in solution. The DLS assays showed that the 10E8 variants each exhibited almost no heterogeneity of population size, indicating that the 10E8 variants were monodisperse in solution. The identified hydrodynamic radius for each variant fell within a normal range.

8. DLS thermal ramping (DLS-Melt) was used to evaluate the colloidal and structural thermal transition points for the 10E8 variants. The onset of aggregation (T_onSet) was within the normal range for each of the 10E8 variants.

9. Isothermal Chemical Denaturation was used to evaluate the intrinsic stability of the 10E8 variants as a function of pH. Each of the 10E8 variants had a pH sensitivity in the normal range.

Table 11. Physical properties of 10E8 variants.

Assay	Attribute	VRC01	10E8v4-100cF	10E8v4-100cW	10E8v4 V5R lOOcF	10E8v4
SEC	Peak (isomer) %	Monomer>95% Aggregate<5%	Peak 1: 4.36% Peak 2: 25.77% Peak 3: 69.87%	Peakl: 11.97% Peak 2: 23.14% Peak 3: 64.88%	Peakl: 7.2% Peak 2: 25.6% Peak 3: 67.2%	Peak 1=72.7% Peak 2=20.3% Peak 3=7.0%

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SEC (FreezeThaw 5x)	Freeze Stress	No significant change	No Significant Change	No significant Change	No significant Change
Appearance	Appearance	Clear, colorless; no visible particles	Clear, colorless; no visible particles	Clear, colorless; no visible particles	Clear, colorless; no visible particles	Clear, colorless; no visible particles
UV-VIS (A280, OD350)	Cone. & optical density	OD350 < 0.05	A280: 0.065 OD350: -0.006	A280: 0.65 OD350: -0.013	A280: 0.74 OD350: 0.0009	A280: 0.77 OD350: 0.02
DSC	Thermal Transition Temperature	T_ml> 65°C	T_ml: 73.4°C	T_ml: 72.8°C	T_ml: 72.9°C	T_ml: 75.2°C
CD	Secondary Structure	Primary Minimum at ~218nm	Min. -218 nm and -230 nm	Min. -218 nm and -230 nm	min. -218 nm and -230 nm	Min. -218 nm and 230 nm,
DLS	Pop. Size and heterogeneity	>95% material is primary population (-56 nm) % Pd < 20	100% material is primary population (-56 nm)	100% material is primary population (-5-6 nm)	99% material is primary population (-56nm)	100% material is primary population (-5-6 nm)
DLS Melt	Colloidal/ Structural Thermal Transition	Γonset > 60°C	T_Onset⁼ 70.2	T_Onset⁼ 70.0	Tonse t= 69.3	To„set=71.5°C
Isothermal Chemical Denaturation	Ci/2> 5.5 at one or more conditions tested	Cm >5.5 (pH>5.5 and 0-200 mM NaCl)	Cm >5.5 (pH >5 .5 and 0-200 mM NaCl)	Cm >5.5 (pH>5.5 and 0200 mM NaCl)	Ci/₂>5.5 (pH>5.5and 0200 mM NaCl)

In view of the above, the 10E8v4, 10E8v4 SlOOcW, 10E8v4 SlOOcF, and 10E8v4 V5R SlOOcF antibodies were found to have substantially similar physical characteristics that fell within a normal range for antibodies that have successfully been produced using large-scale production techniques.

However, additional analysis of antibody solubility and the ability to concentrate the antibody variants revealed that 10E8v4 SlOOcF and 10E8v4 V5R SlOOcF antibodies were superior in some ways to 10E8v4 SlOOcW. In particular, difficulties with concentrating 10E8v4 SlOOcW were observed, with substantial oprecipitation of antibody from solution when trying to reach concentrations of 20 mg/ml. The other three antibodies (10E8v4, 10E8v4 SlOOcF, and 10E8v4 V5R SlOOcF) did not have difficulties reaching 20 mg/ml.

For example, the standard antibody expression and purification protocol used to generate the 10E8v4 variants included transient expression of heavy and light chains in Expi293F cells, purification of secreted antibody from the cell media using a Protein A purification column, elution of the antibody from the column with elution buffer (pH 2.8), buffer exchange into PBS (pH 7.4) by dialysis, and concentration of the antibody solution. For the 10E8v4, 10E8v4 SlOOcF, and 10E8v4 V5R SlOOcF antibodies, yield fell within a normal range of about 70-80% purified concentrated antibody relative to the amount of corresponding antibody eluted from the column. However, yield of 10E8v4 SlOOcW antibody was only about 60% purified concentrated antibody relative to the amount of 10E8v4 SlOOcW antibody eluted from the column. The loss of 10E8v4 SlOOcW antibody was primarily due to precipitation from solution during the concentration step. In contrast, there was minimal loss of antibody from solution during the concentration step for the 10E8v4 SlOOcF and 10E8v4 V5R SlOOcF antibodies.

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Additionally, when the concentration step was conducted using Amicon Stirred Cell Ultrafiltration (nitrogen pressure) in lOmM Citrate-Phosphate, 150mM NaCl pH 6.0 (10E8v4 SlOOcW) or lOmM CitratePhosphate, 150mM NaCl pH 6.5 (10E8v4 SlOOcF and 10E8 V5R SlOOcF)), approximately 5-10% protein loss was observed for 10E8v4 V5R lOOcF and 10E8v4-100cF during the concentration step, but an approximate 40% protein loss was observed for the 10E8v4-100cW variant. The final antibody concentration resulting from these concentration steps also differed substantially between the tested variants. A final concentration of 23-25 mg/mL was achieved for the 10E8v4 SlOOcF and 10E8v4S100cW antibodies. In contrast, a final concentration of about 40 mg/mL was achieved for the 10E8v4 V5R SlOOcF antibody.

To further investigate the effect of the residue present at kabat position 100c of the 10E8 antibody on solubility, 10E8v4 antibodies with SlOOcF, SlOOcH, SlOOcL, SlOOcR, SlOOcW, or SlOOcY substitutions were expressed and purified as discussed above, and assayed for the solubility based on the ability to concentrate (FIG. 24). The antibodies were eluted from the purification column using elution buffer (pH 7.4), dialyzed into PBS (pH 7.4), and 3 ml (0.35 OD) of each variant was concentrated for 30 minutes at 4000 rpm using a 30,000 MWCO concentrator (Amicon Ultra-15 filter MWCO 30,000), in triplicate. As shown in FIG. 24, the 10E8v4 SlOOcW variant was the most difficult to concentrate, and had the greatest final volume of solution following concentration (and the lowest antibody concentration) as assessed by OD280 measurement.

Example 7

Optimized 10E8 antibodies

This example describes mutagenesis of kabat position SlOOc of the 10E8v4 antibody to several different amino acids to evaluate the relative effect of this residue HIV-1 neutralization by the 10E8v4 antibody. 10E8v4 antibodies with SlOOcF, SlOOcH, SlOOcL, SlOOcR, SlOOcW, or SlOOcY substitutions were expressed and purified as discussed in Example 5, and assayed for the HIV-1 neutralization using a pseudovirus assay. As shown in Table 12, 10E8v4 variants with the slOOcF and SlOOcW substitutions showed the greatest improvement in mean neutralization potency over 10E8v4 (—20- and 40-fold lower mean IC50 values relative to 10E8v4). In combination with the findings discussed above, these results show that 10E8v4 variant antibodies that include a SlOOcF substitution possess an optimal combination of HIV-1 neutralization, solubility, and auto-reactivity properties, relative to the many 10E8 variants tested.

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Table 12. HIV-1 neutralization by 10E8v4 variants

IC50 pg/ml
		10E8v4 lOOcF	10E8v4 lOOcH	10E8v4 lOOcL	10E8v4 lOOcR	10E8v4 lOOcW	10E8v4 lOOcY	10E8 v4
Clade	Virus	YDK	YDK	YDK	YDK	YDK	YDK	YDK
A	KER2008.12.SG3	>50	>50	>50	>50	>50	>50	>50
ACD	6095.V1.C10.SG3	0.001	0.011	0.001	0.011	0.001	0.001	0.004
AE	TH966.8.SG3	0.002	0.091	0.011	0.168	0.001	0.011	0.056
B	6101.10.SG3	0.001	0.103	0.007	0.176	0.001	0.009	0.052
B	PVO.04.SG3	0.147	3.400	0.507	6.450	0.088	0.412	3.070
B	YU2.DG.SG3	0.073	2.590	0.333	3.870	0.042	0.376	1.490
C	CNE31.SG3	0.097	2.240	0.354	2.540	0.041	0.368	1.320
C	ZM106.9.SG3	>50	>50	>50	>50	>50	>50	>50
C	ZM215.8.SG3	0.003	0.141	0.015	0.363	0.001	0.028	0.122

	Median IC50	0.003	0.141	0.015	0.363	0.001	0.028	0.122
	Geometric Mean	0.009	0.312	0.033	0.497	0.004	0.037	0.189
	Fold Improvement over 10E8v4	21.7	0.6	5.7	0.4	42.8	5.1	1
IC80	pg/ml
		10E8v4 lOOcF	10E8v4 lOOcH	10E8v4 lOOcL	10E8v4 lOOcR	10E8v4 lOOcW	10E8v4 lOOcY	10E8 v4
clade	virus	YDK	YDK	YDK	YDK	YDK	YDK	YDK
A	KER2008.12.SG3	>50	>50	>50	>50	>50	>50	>50
ACD	6095.V1.C10.SG3	0.001	0.046	0.002	0.048	0.001	0.006	0.015
AE	TH966.8.SG3	0.017	0.488	0.079	0.648	0.007	0.075	0.291
B	6101.10.SG3	0.008	0.643	0.049	0.891	0.006	0.063	0.263
B	PVO.04.SG3	0.696	14.000	2.210	25.000	0.388	3.440	11.90 0
B	YU2.DG.SG3	0.482	10.900	1.750	12.000	0.331	2.250	6.330
C	CNE31.SG3	0.306	6.790	1.010	8.420	0.149	1.390	4.040
C	ZM106.9.SG3	>50	>50	>50	>50	>50	>50	>50
C	ZM215.8.SG3	0.020	0.966	0.095	1.340	0.012	0.129	0.502

	Median IC80	0.020	0.966	0.095	1.340	0.012	0.129	0.502
	Geometric Mean	0.040	1.464	0.162	1.913	0.025	0.235	0.780
	Fold Improvement over 10E8v4	19.4	0.5	4.8	0.4	31.4	3.3	1.0

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It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described embodiments.

Claims

We claim all such modifications and variations that fall within the scope and spirit of the claims below.

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It is claimed:

1. An isolated antibody, comprising:

a heavy chain variable region (Vh) comprising a heavy chain complementarity determining region (HCDR)l, a HCDR2, and a HCDR3 of the Vh set forth as SEQ ID NO: 75, and a light chain variable region (Vl) comprising a light chain complementarity determining region (LCDR)l, a LCDR2, and a LCDR3 of the V_L set forth as SEQ ID NO: 6 (10E8v4 SlOOcF CDRs); and wherein the antibody specifically binds to gp41 and neutralizes HIV-1.
2. The isolated antibody of claim 1, wherein the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences set forth as: SEQ ID NOs: 7, 8, 77, 10, 11, and 12, respectively (10E8v4 SlOOcF IMGT CDRs).
3. The isolated antibody of claim 1 or claim 2, wherein the Vh comprises arginine, glutamate, serine, lysine, aspartate, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82c, 84, 87, 89, 105, and 110, respectively; and the Vl comprises alanine, serine, aspartate, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively.
4. The isolated antibody of any of claims 1-3, comprising an arginine residue at kabat position
5.

5. The isolated antibody of any of claims 1-4, wherein the Vh and the Vl comprise amino acid sequences at least 90% identical to SEQ ID NOs: 75 and 6, respectively (10E8v4 SlOOcF).
6. The isolated antibody of any of claims 1-4, wherein the Vh and the Vl comprise amino acid sequences at least 90% identical to SEQ ID NOs: 76 and 6, respectively (10E8v4 V5R SlOOcF).
7. The isolated antibody of claim 1, wherein the Vh and Vl comprise the amino acid sequences set forth as SEQ ID NOs: 75 and 6, respectively (10E8v4 SlOOcF).
8. The isolated antibody of claim 1, wherein the Vh and Vl comprise the amino acid sequences set forth as SEQ ID NOs: 76 and 6, respectively (10E8v4 V5R SlOOcF).

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9. An isolated antibody comprising:

a heavy chain variable region (Vh) comprising a heavy chain complementarity determining region (HCDR)l, a HCDR2, and a HCDR3 of the Vh set forth as SEQ ID NO: 5, and a light chain variable region (Vl) comprising a light chain complementarity determining region (LCDR)l, a LCDR2, and a LCDR3 of the Vl set forth as SEQ ID NO: 6 (10E8v4 CDRs), wherein the Vh comprises arginine, glutamate, serine, lysine, aspartate, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively; and the Vl comprises alanine, serine, aspartate, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamate, and aspartate residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively; and wherein the antibody specifically binds to gp41 and neutralizes HIV-1.
10. The isolated antibody of claim 9, wherein the Vh and the Vl comprise amino acid sequences at least 90% identical to SEQ ID NOs: 5 and 6, respectively (10E8v4).
11. The isolated antibody of claim 9, wherein the Vh and Vl comprise the amino acid sequences set forth as SEQ ID NOs: 5 and 6, respectively (10E8v4).
12. The isolated antibody of any of claims 1-8, wherein (a) the V_H further comprises one of a E1R, E1W, G15R, S25W, D28W, D30W, D30R, N31W, N31R, P52bR, D72R, or E81R substitution (kabat numbering) relative to SEQ ID NO: 76;

(b) the Vh further comprises D72R, E1R D28W, and P52bR substitutions (kabat numbering) relative to SEQ ID NO: 76;

(c) the Vh further comprises a D28Y substitution (kabat numbering) relative to SEQ ID NO: 76;

(d) the V_L further comprises one of a D7W, A14R, Q17R, T18R, T20R, D26R, K51R, N53R, S65R, G68R, G77R, or G100R substitution (kabat numbering) relative to SEQ ID NO: 6;

(e) the Vl further comprises D7W, D26R, and G68R substitutions (kabat numbering) relative to SEQ ID NO: 6;

(f) the Vh further comprises D72R, E1R D28W, and P52bR substitutions (kabat numbering) relative to SEQ ID NO: 76, and the Vl further comprises D7W, D26R, and G68R substitutions (kabat numbering) relative to SEQ ID NO: 6;

(g) the Vh further comprises a P52bR substitution (kabat numbering) relative to SEQ ID NO: 76, and the Vl further comprises D7W and G68R substitutions (kabat numbering) relative to SEQ ID NO: 6; or (h) the Vh and Vl further comprise one of the following pairs of substitution relative to SEQ ID NOs: 76 and 6, respectively: E1R and D7W; E1R and D26R; E1R and G68R; D28W and D7W; D28W and D26R; D28W and G68R; D28W and Q17R; D28W and T18R; D28W and T20R; D28W and K51R; D28W

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P52bR and T20R; P52bR and K51R; P52bR and G77R; D72R and D7W; D72R and D26R; or D72R and

G68R; and wherein the antibody specifically binds to gp41 and neutralizes HIV-1.
13. The isolated antibody of any of the prior claims, wherein the antibody dissolves to a concentration of at least 5 mg/ml in phosphate buffered saline, pH 7.4, at 20°C.
14. The isolated antibody of any of the prior claims, wherein the antibody neutralizes at least

95% of the viral strains listed in FIG. 16 with an IC50 of 50 Ug/ml or less.
15. The isolated antibody of any of the prior claims, comprising human framework regions.
16. The isolated antibody of any of the prior claims, comprising a human constant region.
17. The isolated antibody of any of the prior claims, wherein the antibody is an IgG, IgM or

IgA.
18. The isolated antibody of any of the prior claims, comprising a recombinant constant region comprising a modification that increases binding to the neonatal Fc receptor.
19. The isolated antibody of claim 18, wherein the antibody is an IgGl and the modification that increases binding to the neonatal Fc receptor comprises M428L and N434S amino acid substitutions.
20. An antigen binding fragment of the antibody of any of claims 1-19.
21. The antigen binding fragment of claim 20, wherein the antigen binding fragment is a Fv,

Fab, F(ab')2, scFV or a SCFV2 fragment.
22. A bispecific antibody comprising the antibody or antigen binding fragment of any of the preceding claims.
23. The antibody or antigen binding fragment of any of the prior claims, linked to an effector molecule or a detectable marker.
24. The antibody or antigen binding fragment of claim 23, wherein the detectable marker is a fluorescent, enzymatic, or radioactive marker.

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25. An isolated nucleic acid molecule encoding the antibody or antigen binding fragment of any of claims 1-24.
26. An isolated nucleic acid molecule encoding the Vh, the Vl, or the Vh and Vl, of the antibody or antigen binding fragment of any of claims 1-24.
27. The nucleic acid molecule of any one of claims 25-26, wherein:

the Vh of the antibody or antigen binding fragment comprises the nucleic acid sequence set forth as SEQ ID NO: 45 (H6-51 l-4mut), SEQ ID NO: 78 (H6-51 l-4mut SlOOcF), or SEQ ID NO: 79 (H6-51 l-4mut V5R SlOOcF); or the Vl of the antibody or antigen binding fragment comprises the nucleic acid sequence set forth as SEQ ID NO: 47 (rL3-6mut); or the Vh and the VLof the antibody or antigen binding fragment comprise the nucleic acid sequence set forth as SEQ ID NOs: 45 and 47, respectively (10E8v4), SEQ ID NOs: 78 and 47, respectively (10E8v4 SlOOcF), SEQ ID NOs: 79 and 47, respectively (10E8v4 V5R, SlOOcF).
28. The nucleic acid molecule of any of claims 25-27, operably linked to a promoter.
29. An expression vector comprising the nucleic acid molecule of any of claims 25-28.
30. A host cell comprising the nucleic acid molecule of any one of claims 25-29, particularly wherein the host cell is a eukaryotic host cell such as a human host cell.
31. A pharmaceutical composition for use in treating an HIV-1 infection, comprising:

a therapeutically effective amount of the antibody, antigen binding fragment, nucleic acid molecule, or expression vector of any of claims 1-29; and a pharmaceutically acceptable carrier.
32. A method of producing an antibody or antigen binding fragment that specifically binds to gp41, comprising:

incubating the host cell of claim 30 under in vitro conditions sufficient for expression of the nucleic acid molecule or expression vector to produce the antibody or antigen binding fragment; and purifying the antibody or antigen binding fragment.
33. A method of detecting an HIV-1 infection in a subject, comprising:

-107WO 2017/079479

PCT/US2016/060390 contacting a biological sample from the subject with the antibody or antigen binding fragment of any of claims 1-24 under conditions sufficient to form an immune complex; and detecting the presence of the immune complex on the sample, wherein the presence of the immune complex on the sample indicates that the subject has the HIV-1 infection.
34. A method of inhibiting or treating an HIV-1 infection in a subject, comprising administering to the subject a therapeutically effective amount of the antibody, antigen binding fragment, nucleic acid molecule, or expression vector of any of claims 1-29, thereby preventing or treating the HIV-1 infection.
35. The method of claim 34, wherein the subject is at risk of an HIV-1 infection.
36. The method of claim 34, wherein the subject has an HIV- infection
37. The method of claim 36, wherein the subject has AIDS.
38. The method of any of claims 34-37, further comprising administering to the subject an additional antibody, antigen binding fragment, or nucleic acid encoding the additional antibody or antigen binding fragment, wherein the additional antibody or antigen binding fragment specifically binds to HIV-1 Env and neutralizes HIV-1 infection.
39. The method of claim 38, wherein the additional antibody is a VRCOl-class antibody.
40. The method of claim 39, wherein the VRCOl-class antibody is VRC01 or VRC07-523.

41. Use of the antibody, antigen binding fragment, nucleic acid molecule, expression vector, or pharmaceutical composition of any of claims 1-29 or 31 to inhibit or treat HIV-1 infection in a subject.

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Kabat numbering, IMGT CDRs

ω ω ω ω ω ω ι ω ω ω ω ω ω ι > > > > > > ι o — Η Η Η Η Η Η 1 r-1 > > > > > > r-1 h3 Ω Ω Ω Ω Ω Η Η Η Η Η Η Χι Ο Ο Ο Ο Ο Ο 1 cd Ω Ω σ οι οι 1 Ο Ο Ο ο ο Ο 1 CS Ω Ω Ω 5 1 > > > > > > 1 oo oo oo oo oo oo 1 Ω Ω Ω Ω Cs Cs 1 Q Ω Ω Ω Ω Ω 1 O O O O O O CN Ο Ο Ο Ο) Ο) Ο) 1 ω ω ω ω ω ω cd H h Ω Ω Ω Ω Ω 1 ϋ — o o o o o o Q Λ! >Η Ω Ω Ω Ω Ω 1 Λ — Ω CM CM CM CM CM o m — ω ω Ω Ω Ω Ω 1 nJ — o o o o o o 1 H ω ω Ω Ω Ω Ω 1 Ol — H H H w w H 1 ο ο Ο Ο Ο Ο 1 m 1—1 1—1 1—1 1—1 1—1 1—1 1 tP — CM Ω Ω Ω Ω Ω 1 (H CM Ω Ω Ω Ω Ω 1 (U —· >Η Ω Ω Ω Ω Ω 00 o — Ω cd cd Cd Cd cd 1 Ό ο ο Ο Ο Ο Ο Ω m o o o o o o 1 ϋ —· ω ω ω ω ω ω Ω > > > > > > 1 Λ — Ω Ω Ω Ω Ο Ω Ω is is 1 nJ — 34 Ω Ω Ω Ω Ω 1 ω ω ω ω ω ω 1 o — Q Ω Ω Ω Ω Ω 1 j ω Ω Ω Ω Ω 1 o >Η Ω Ω Ω Ω Ω 1 o o o o o o 1 r-1 >Η Ω Ω S S Ω 1 Cd Cd Cd Cd Cd Cd CN Cd Ω Ω Ω Ω Ω 1 o o o o o o cd ο Ο Ο Ο Ο Ο 1 cm CM CM CM CM CM Ω Η Η Η Η Η Η 1 o — CM CM CM CM CM CM 1 cd Ω Ω Ω Ω Ω 1 Oi o o o o o 1 >< >< >< >< >< < 1 cd cd cd cd cd cd 1 > > > > > > 1 Ω Ω is is 1 ο Ο Ο Ο Ο Ο 1 H H H H H H 1 34 Ω Ω Ω Ω Ω 1 S s s s s s 1 o >Η Ω Ω Ω Ω Ω 1 σ> η3 Ω Ω >Η >Η >Η 1 Ο Ο Ο Ο Ο Ο 1 \S \s \s \s \s \s 1 ω ω ω Η Η Η 1 >< >< >< >< >< >< 1 Q Ω Ω Ω Ω Ω 1 Ω Ω Ω Q Q Ω C—1 33 Ω Ω Ω Ω Ω 1 o — Ω Ω Ω Ω Ω Q cd S S Η Η Η Η 1 ω Ω Ω Ω Ω Ω Ω Q cd Ω Ω Ω Ω Ω 1 Ω Ω Ω Q o ϋ —· J Ω Ω > > > 1 Ω Ω Ω Ω Ω Ω 1 Λ Ω Ω Ω Ω Ω 1 O o O o o O 1 nJ Ω Ω Ω Ω Ω 1 οι S S S S S S 1 00 33 Ω Ω Ω Ω Ω 1 oo oo oo oo oo oo 1 o — η3 Ω Ω Ω Ω Ω 1 >< >< >< >< >< >< 1 00 >η Ω Ω Ω Ω Ω 1 oo oo oo oo oo oo 1 η3 Ω Ω Ω Ω Ω 1 o o o o o o 1 34 Ω Η Ω Η Η co oo oo oo oo oo oo 1 Ω Ω Ω Ω Ω o — Ω Ω Ω Ω Ω Ω 1 1—I 1—I Ω 1—I Ω Ω Χι Ol cd cd cd cd cd cd 1 ω Ω Ω ω Η Η 1 Ω Ω Ω Ω Ω Ω 1 Ω Ω Ω Ω Ω 1 oo oo oo oo oo oo 1 J Ω Ω Ω Ω Ω 1 O O O O O O 1 cd Ω Ω Ω Ω Ω 1 O O O O O O 1 o —· ω ω ω ω ω ω ι CM CM CM CM CM CM 1 1—I 1—I 1—I 1—I 1—I ι—ι ι Cd Cd Cd Cd Cd Cd C—1 Η Η Η Η Η Η ι > > > > > > cd 34 Ω Ω Ω Ω Ω 1 Ω Ω Ω Ω Ω Ω h cd Ω Ω Ω Ω Ω 1 o — o o O O O O 1 ο Ο Ο Ο Ο Ο 1 r-1 o o O O O O 1 33 Ω Ω Ω Ω « 1 o o O O O O 1 > > > > > > ι oo oo oo oo oo oo 1 CM Ω Ω ω ω ω ι ω ω ω ω ω ω 1 >< >< >< W W W ι > C C > > > 1 o — >< >< >< >< >< < ι ω ω Ω Ω Ω Ω 1 co >Η Ω Ω Ω Ω Ω 1 Oi Pi Pi Pi Pi Pi 1 Ω Ω Ω Ω Ω Ω 1 > > > > > > 1 r-1 — ω ω ω ω ω ω 1 T—1 τ—1 T—1 +j τ—1 μ LO LO £ £ Ω >H Ω >Η H 1 Η 1 Ω r-- T—1 Ω τ—1 Ω oo ω T—1 r-1 ω ω τ—1 Ω 00 1 LO m H 00 I LO m Η ω CD 1 o 33 CD ι Ο o O O CD CO s ο Ο ο CD <ο S T—1 cc X! 33 s 1—1 τ—1 Ω X Ω S Η

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FIG. IB

π> — Ω Ω Ω I <£> — > > > 1 ο Η Η Η 1 CN Γ-) Ω Ω Ω 1 Ω Ω Ω Ω Ω Ω Ω Ω Η Η Η Ω ο — ο Ο Ο Ο Ο Ο Ο Ω m ο — Ο Ο Ο 1 ο Ο Ο Ο 1 >Η Ω Ω 1 Γ-) 1+ 1+ 1+ 1 1+ 1+ 1+ 1 Ω Ω 1 Ω Ω 1 > > > 1 1—I > > 1 ω ω ω 1 Λ Ω Ω 1 υ — Ω Ω Ω 1 >< >< >< CN Λ — Ω Ω Ω 1 Ο) Ο) Ο) Ω π> — ω ω ω CO Ο ο ο 1+ m — Ο Ο Ο Ω ο — Λ Ω Ω 1 σ> ω ω ω Ω Ω Ω 1 Ω Ω Ω Ο Ω Ω 1 Ω Ω Ω 1 Ο) Ο) Ο) 1 Ω Ω Ω 1 >Η Ω Ω 1 ο — ω ω ω 1 Ω 1 σ> ω ω ω 1 (0 ω ω ω 1 Oh >< >< >< 1 Q Ο Ο Ο 1 o Ω Ω Ω 1 >Η Ω Ω 1 Ω Ω Ω 1 Eh Ω Ω τ—1 ω Ω Ω 1 0 ο — ω ω ω Ω >< >< >< 1 2 ω C4 Ω Ω Ω Ω W W 1 H Ω Ω Ο Ω Ω Ω 1 ω ω ω 1 ω ω ω 1 ο — >< >< >< 1 00 Ο) Ο) Ο) 1 β Ω Ω Ω 1 >< >< >< 1 ^arH Ο Ο Ο 1 ο ο ο 1 μ Ω Ω Ω 1 ω Fi Fi 1 (1) ο ο ο 1 1—I 1—I 1—I 1 η Η Η Η 1 Η Η Η 1 Έ 1—I 1—I 1—I 1 Ω Ω Ω 1 ►Η β ο — Η Η Η 1 ω ω ω 1 β m > > > 1 >< >< >< CO Η Η Η 1 ο — Ω Ω Ω Ω 1) Ω Ω σ 1 Ω Ω Ω Ω (0 Ο Ο 1 Ο Ο Ο 1 43 Ω Ω Ω 1 ω ω ω 1 (0 >< >< >< τ—1 >< >< >< 1 ki > > > Ω ω ω ω 1 ω ω ω 1+ Ο Ο Ο 1 A > > > 1 ω ω ω 1 > ο — . 1 1+ 1+ 1+ 1 γ-) Ο Ο C 1 Ω Ω Ω 1 Η Η CM 1 ο — Ω Ω Ω 1 ω Ω Ω 1 <£> Ω Ω Ω 1 Ο) Ο) Ο) 1 > Η Η 1 Η Η Η 1 Ο Ο Ο 1 Ω Ω Ω 1 ω ω ω 1 ω ω ω 1 Ω Ω Ω 1 >Η Ω ω 1 Ω Ω Ω 1 Γ-) — ω ω <! 1 Ω Ω Ω 1

+J <x>

CO

J

So

H

O s

I—I +J <£>

ω co o J

T—I (-)

H o S I—I

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FIG. 1C

Ο ο ο ο ο ο I ω ω ω Ω Ω Ω I > > > > > > 1 λ ω ω ω ω ω 1 >< >< >< W W W 1 Ο — >< >< >< >< >< >< 1 <£> >Η Ω Ω Ω Ω Ω 1 Ω Ω Ω Ω Ω Ω 1 ω ω ω ω ω ω 1 > > > > > > CN ω ω ω ω ω ω 1 ω ω ω ω ω ω (X > > > > > > 1 !S !S Ω Ω Ω Ω Ω ο —· Η Η Η Η Η Η 1 Ο Ο Ο Ο Ο Ο ο γ-1 > > > > > > ω ω ω ω ω ω 1 γ-1 Ω (A ϋ — ο ο ο ο ο ο 1 Η Η Η Η Η Η to X) — ω ω ω ω ω ω 1 Ο Ο Ο Ο Ο Ο 1 π> — ο ο ο ο ο ο 1 (Α (Α (Α σ οι οι 1 d — Η Η Η ω ω Η 1 Ο Ο Ο ο ο ο 1 m I—1 1—1 1—1 1—1 1—1 1—1 1 1 ο - (X (X (X (X (X (X 1 m Q Q Q Q Q Q 1 Ο Ο Ο Ο Ο Ο 1 Ο) Ο) Ο) Ο) Ο Ο 1 > > > > > > 1 Η to to to to to to 1 1 Λ! >Η >Η >Η >Η >Η >Η 1 ω ω ω ω ω ω 1 ΓΊ — ω ω ω ω ω ω 1 ω ω ω ω ω ω 1 Η - ω ω ω ω ω ω 1 ο ο ο ο ο ο 1 Λ — ο ο ο ο ο ο 1 Ω Ω Ω Ω Ω Ω CN tP λ λ λ λ λ λ 1 (0 ο ο ο ο ο ο (X Ή — λ λ λ λ λ λ CO erf ω ω ω ω ω ω Ω ω >Η >Η >Η >Η >Η >Η (A Q ο — ω ω ω ω ω ω 1 τ) ο ο ο ο ο ο Q Γ ) Ο) Ο) Ο) Ο) Ο) Ο) 1 υ — ω ω ω ω ω ω o (Α (Α (Α (Α (Α (Α 1 Λ — 1 > > > > > > 1 π> to to to to to to 1 (0 1 ο — Q Q Q Q Q Q 1 _Q ο >Η >Η >Η >Η >Η >Η 1 rM (0 γ-1 >Η >Η >Η S S >Η 1 _U Η Η Η Η Η Η 1 1 S S S S S S τ— Ο Ο Ο ο ο Ο 1 (X Η Η Η Η Η Η 1 ϋ' >< >< >< >< >< >< Ω Ω Ω Q Ω Ω Ο M Ή (4 (4 (4 (4 (4 (4 1 μ C C C C C C 1 Φ η ο — Ω Ω Ω Ω Ω Ω 1 Ο Ο Ο ο ο Ο 1 ω Ω Ω Ω Ω Ω Ω 1 to to to to to to 1 Έ Ω Ω Ω Ω 1 Ο >Η >Η >Η >Η >Η >Η 1 R Ω Ω Ω Ω Ω Ω 1 σ> >Η >Η >Η 1 ι-^ ο ο ο ο ο ο 1 Ο Ο Ο ο ο Ο 1 Μ ω ω ω ω ω ω 1 ω ω ω Fi Fi Fi 1 >< >< >< >< >< >< 1 Q Q Q Q Q Q 1 (0 ω ω ω ω ω ω 1 ω ω ω ω ω ω 1 η ο ο ο ο ο ο 1 S S Fi Fi Fi Fi 1 (0 ω ω ω ω ω ω 1 (Α (Α (Α (Α (Α (Α 1 ο d — Ω (X Ω (X Ω (X Ω (X Ω (X Ω (X 1 1 ϋ Λ j s j s 1 1 κ® Ω Ω Ω Ω Ω Ω 1 π> s s CO > ω ω ω ω ω ω 1 d —· S S S S S S (A Ο Ο Ο Ο Ο Ο 1 00 ω ω ω ω ω ω to Ο Ο Ο Ο Ο Ο 1 ο j j j j j j 1 Ω Ω Ω Ω Ω Ω 1 00 >Η >Η >Η >Η >Η >Η 1 Ω Ω Ω Ω Ω Ω τ— j j j j j j 1 > > > > > > (X to to Fi to Fi Fi 1 Ω Ω Ω Ω Ω Ω Ω 1 ο — Ο Ο Ο Ο Ο Ο 1 1—I 1—I 1—I 1 γ-1 ο ο ο ο ο ο 1 ω * * ω Fi Fi 1 ο ο ο ο ο ο 1 1 ω ω ω ω ω ω 1 Ω Ω Ω 1 ω ω ω ω ω ω 1 (Α (Α (Α (Α (Α (Α 1 > ι< ι< > > > 1 ο — ω ω ω ω ω ω 1 ω Ω Ω ω ω ω 1 1—I 1—I 1—I 1—I 1—I 1—I 1 Ο) Pi Pi Pi Pi Pi 1 Η Η Η Η Η Η 1 > > > > > > 1 to to to to to to 1 γ-1 — ω ω ω ω ω ω 1 (Α (Α (Α (Α (Α (Α 1

v—1 τ—1 v—1 +J τ—1 μ LO LO £ £ γ H 1 Η 1 r- Γ- T-1 t—1 Γ- Γ- Τ-1 γΗ ω ω v—1 r-1 -P ω ω Τ—1 γ-1 -Ρ CO 1 LO m (ύ CO I LO m (ύ ω <D <D 1 1 Λ ω LD LD 1 I Λ o O O <D LD <£> (ύ ο Ο ο LD LD <£> (ύ v—1 X! X X X S Λί Τ—1 X X X X S Ρί

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ω ω ω 1 CL CL CL 1 cL CL CL CN S S S CL S S S Q ni — j J 1 +: +: +: O co — > > J 1 O — o o o 1 o H H > 1 m r-1 J J H 1 +: +: J 1 H H +: CL +1 +1 +1 1 o o H +1 J J J 1 o — o o o 1 J J J 1 o o o o 1 1—1 > > 1 r-1 +1 +1 o 1 CL CL CL 1 +1 >< C C CN Oi o o CL > > 1 O o o +1 ω ω > 1 o — CL CL CL 1 ϋ — J J ω 1 +: +: +: 1 44 — CL CL J 1 +: +: +: 1 ni — ω ω CL CO Oi o o 1 m — O O ω CL 1 σ> ω ω O Q s s s 1 +: +: ω o 10 Q Q +: 1 Pi CL CL Q 1 Q ω ω ω 1 o — ω ω CL 1 O C C C 1 σ> ω ω ω 1 1 ω +J CL CL CL 1 nJ o — ω ω ω c—1 Ο Ο 1 44 co CL CL CL CL Ο 1 nJ J J J Q 1 44 ω ω ω O ω Q 1 Q Q Q 1 c C Q 1 O O O 1 Q w C 1 tP CL CL CL 1 Q Q w 1 G ω ω Q 1 H o — c c ω 1 M O O O 1 00 o o c 1 <D H H H 1 c c o 1 -9 1—1 1—1 1—1 1 o o c 1 ε o — H H H 1 ω H o 1 G OI > > > 1 1—1 1—1 H 1 G H H H 1 H H 1—1 1 CL CL oi 1 J J H CO +J O O ¢4 1 ω ω J CL ni J J j 1 C C ω +1 44 C C c 1 o — CL CL C 1 ni > > > c—1 S S CL 1 ¢4 ω ω ω CL O O S 1 > > > +1 ω ω O 1 κβ o 1 C C ω 1 r-1 o o pjj 1 ω ω C 1 H H CM 1 O O ω 1 ω Q Q 1 ω ω O 1 o O O 1 +1 +1 ω 1 H H H 1 CL CL +1 1 J J J 1 o — Q Q CL 1 ω ω ω 1 co CL CL Q 1 w 1 > H CL 1 r-1 — ω ω <! 1 O O H 1 O

+J +J H G H G τ ε τ ε co co 00 | 00 | ω η co ω η ω C L hl C L |4 t—I + M t—I + M

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FIG. IE

ω ω ω ω i ω ω ω ω 1 > > > > 1 o — H Η Η Η 1 rH > > > > rH A A A A cP H Η Η Η A O Ο Ο Ο 1 CP σ σ σ 1 > > > > 1 O ο ο ο 1 ω ω ω ω 1 A A A A 1 A S A A 1 O O O O CN a A A A cP Q Ω Q Q 1 ϋ o O O O Q Oi Ο) Ο) Ο 1 43 a A A A O H A A A A 1 nJ — o O O O 1 Λ! — A A A A 1 CN — H H H H 1 n — A A A A 1 m 1—1 1—1 1—1 1—1 1 rl — A A A A 1 41 — O O O Ο 1 tP A A A A 1 o CP CP CP CP 1 m A A A A 1 m o O O O 1 <U — A A A A 00 > > > > 1 Ό — O O O O cP A A A A 1 υ — ω ω Al Al Q a A A A 1 43 — A A A A o j A A A 1 nJ — A A A A 1 o O O O 1 o — Q Ω Q Q 1 A A A A CN o A A A A 1 o O O O cP rH A A A A 1 a A A A A A A A A 1 o a A A A 1 O O O O 1 10 Oi a a a 1 H H H H 1 Ci cP CP CP CP 1 CP CP CP CP 1 Q > > > > 1 >< >< >< >< 1 O A A A A 1 H H H H 1 EH S S s s 1 O O O O 1 0 A A A A 1 2 O - A A A A 1 H \S \S A A 1 σ> A A X X 1 >< >< >< >< 1 O o o o 1 A A A A C—1 ω Fi Fi Fi 1 tp o — Q Q Q Q cP Q Ω Q Q 1 c ω IA A A A Q A A A A 1 H Q Q Q Q O S Fi Fi Fi 1 μ A A A A 1 CP cP cP cP 1 <U 43 O O O O 1 υ — A > > > 1 43 — A A A A 1 nJ A A A A 1 3 ω ω ω ω 1 CN S S S S 1 a >< >< >< >< 1 00 A A A A 1 ω ω ω ω 1 o — A A A A 1 +J o o o o 1 00 A A A A 1 nJ ω ω ω ω 1 A A A A 1 4J o A A A A 1 A Fi Fi Fi 00 nJ CN cP cP cP cP 1 A A A A cP X A A A A 1 1—1 A X A A ω ω ω ω 1 ω Fi Fi Fi 1 .a O O O O 1 A A A A 1 > O O O O 1 A Q Q Q 1 A A A A 1 CP cP cP cP 1 A A A A C—1 o — ω ω ω ω 1 > > > > cP 1—1 1—1 1—1 1—1 1 A A A A A H H H H 1 o —· O O O O 1 A A A A 1 rH O O O O 1 cP cP cP cP 1 O O O O 1 o o o o 1 ω ω ω ω 1 A A X A 1 A A A A 1 > > > > 1 > > > Cil 1 A w w w 1 A A A A 1 >< w A w 1 a Ci Ci Ci 1 o — >< >< >< >< 1 > > > > 1 co A A A A 1 rH A A A A 1 Q Q Q Q 1 A A υ υ o o o o A C—1 A c—1 υ ω υ ω o o o o C—1 LO c—1 LO ω > ω > H H A A > > > > > > 00 00 00 00 H 00 00 00 00 H A A H H O A A H H o o o o o s o o o o s C—1 C—1 C—1 C—1 1—1 c—1 c—1 c—1 c—1 1—1

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di — J J 1 <£> — > > 1 o H H 1 CM r-1 J J 1 (X Q H H (X o — o O O O O IA m o O O 1 o O O 1 >H 1 r-1 IA IA 1 IA IA 1 J J 1 J J 1 > > 1 1—1 > 1 co co 1 Λ Λ 1 ϋ — J J 1 >< >< CN 44 — (X (X 1 O O (X di co co CO o o IA m — O O (X o — Λ Λ 1 σ> co co Q 1 o 1 Q Q 1 Oi o 1 (X (X 1 1 o —· co co 1 1 σ> co co 1 co co 1 ΙΟ >< >< 1 Fi Ο Ο 1 ο 1 2 1 1 H K K I—1 ω Q 1 o — co co (X >< >< 1 ω (X (X Q Q w 1 tP J J O Q Q 1 c co co 1 ω ω 1 LL H o — >< >< 1 1 M 00 o o 1 <D Q Q 1 >< >< 1 • -9 O O 1 O O 1 ο E (X (X 1 co Fi 1 3 O O 1 1—1 1—1 1 LL G H H 1 H H 1 1—1 1—1 1 J J 1 P o — H H 1 co co 1 di OI > > 1 >< >< CO 44 H H 1 o —· (X (X (X di kJ (X oi 1 s S IA HM O 1 o o 1 J J j 1 co co 1 > >< >< I—1 >< >< 1 > > (X co co 1 co co IA O O 1 > > 1 co co 1 o — . * 1 ΙΑ ΙΑ 1 r-1 o C 1 (X (X 1 H CM 1 o Q Q 1 ω Q 1 <£> Λ Λ 1 o O 1 > H 1 H H 1 O O 1 J J 1 co co 1 ω ω 1 Λ Λ 1 w 1 (X (X 1 r-1 — co <! 1 S S 1

H Η > > CO CO H CO CO Η ω ω ο ω ω ο ο o s ο ο s ι—1 ι—1 1—1 ι—1 ι—1 1—1

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FIG. 2

Turbidity at 1X PBS pH 7.4 G.8-,...................................................

H m _1 + > st 74Y-511 + > CO CO 00 ID CD LLl O r—1 U ΞΕ <_) ΞΕ

•As\\\X /:+0% >S$W: /OO

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FIG. 3

IC80 pg/ml

HC6-S74Y- 511 + rL3 HC6-S74Y + rL3 10E8 WT virus Q23.17.SG3 3.900 1.390 5.020 UG037.8.SG3 0.359 0.187 1.030 AC10.29.SG3 0.224 0.217 0.699 JRFL.JB.SG3 2.020 0.713 2.070 DU 151.02.SG3 1.260 1.180 3.100 ZM53.12.SG3 6.600 4.710 12.000 ZM106.9.SG3 50.000 50.000 50.000 TV1.29.SG3 0.401 0.450 1.190 Median IC80 1.640 0.947 2.585 Geometric Mean 1.790 1.224 3.368

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FIG.4A

Turbidity in PBS

FIG.4B

!C50 pg/ml ciade virus A KER2GG8.12.SG3 ACD 6095.V1.C10.SG3 AE TH966.8.SG3 B 6101.10,5G3 B PVO.04.SG3 B YU2.OG.SG3 C CNE31.SG3 C ZM106.9.SG3 C ZM215.8.SG3 Geometric Mean

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FIG. 5

MPER peptide

Heavy chain

Light chain

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FIG. 6

Turbidity at 1X PBS (by dilution)

0.8

0.6s c

o

KO co

G

O

0.40.20.0 r ·τ a/*

0?

nS>

“i-r* fc. b to Λ <b

1. H6-511 V_H +rL3 V_L

2. H6-511-H98Y V_H + rL3 V_L

3. H6-511-H98Y-N28D V_H + rL3 V_L

4. H6-511-H98Y-D31N V_H + rL3 V_L

5. H6-511-H98Y-S52T V_H + rL3 V_L

6. H6-511-H98Y-N28D-D31N V_H + rL3 V_L

7. H6-511-H98Y-N28D-D31N-S52T V_H + rL3 V_L

8. H6-520-H98Y-N28D-D31N-S52T V_H + rL3 V_L

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FIG. 7 dade virus

1Q£8H6 51 /L3

YDK

10£8H6511 _H98Y_N28O J331N_S52T/ 13

YOK

10E8_H6_520 _H98YJM2SD D31N 552T/ L3

YDK

10E8 lOESwt

YOK

A

ACD

AE

B

KER200S,12,5G3

6095.V1.C10.SG3

TH966.8.SG3

6101.10.SG3

PVO.04.SG3

YU2.DG.SG3

CNE31.SG3

ZM106.9.SG3

ZM215.8.S63

1:72:88 0.943 0.287 1.90 3.20 2.04 lllllllll 2.80 1.99 IlllOll! 0,584 1.73 >50 >50 >50 >25

1. H6-511 V_H + rL3 V_L

7. H6-511-N28D-D31N-S52T-H98Y V_H + rL3 V_L

8. H6-520-N28D-D31N-S52T-H98Y V_H +rL3 V_L

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FIG. 8

Turbidity at 1X PBS

FIG. 9

Turbidity at 1X PBS (by dialysis)

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FIG. 10

Turbidity in PBS

1. H6-511-4mut V_H + rl_3 V_L

2. H6-511-4mut V_H + rL3-6mut V_L

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10E8H6 511 /13

FIG. 11

IC5O(pg/ml) ciade virus

A K£R2008,12.5G3

ACD 6095.VX.C1G.5G3

AE TH966.8.SG3

B 6101.10.SG3 lOESwt >50 >50 >50 >25

B PVO.G4.SG3 lllll/lll! 0.943 1.10 1.90 B YU2.DG.SG3 /11/11111 iiiiiiii 2.03 2.80 C CNE31.5G3 11111111 1.68 LOS 1.73 C ZM106.9.SG3 >50 >50 >50 >25

ZM215.8.SG3

Geometric Mean

IC80 (Rg/ml)

Q.125 0.072 0.073 0.140

1. H6-511-4mut V_H + rl_3 V_L

2. H6-511-4mut V_H + rL3-6mut V_L

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FIG. 12

HC6-S74Y/rL3

Potent

Aggregation-prone

Somatic

Variant

H6/L10

Less Potent, but less aggregation-prone

Hydrophobic aa replaced with hydrophilic

Variant 1

HC6-S74Y-511/rL3

Potency retained Solubility improved

H6-511/L10

Less Potent, Solubility improved

4 mutations in V_H + rL3 light chain _ 6 aa changes in rL3 light chain

Variant 3 H6-511-4mut/rL3

Potency retained Solubility improved

Variant 5

HC6-S74Y-511 /rL3-6mut

Potency retained Solubility greatly improved

Variant 4

H6-511 -4mut/rL3-6mut

Potency retained Solubility greatly improved

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FIG. 13A

FIG. 13B

Volume after centrifugation

3000-1 ;2000

JV JS A** ν'* <> OU XT

X

FIG. 13C

Protein oncentration after centrifugation (OD) <8>

&

a*

J*

W/’ -Ucjp·

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FIG. 14

Meas 1 o

o

X © } ©

Φ Φ co co v Φ © t 9 Φ Q_ : τ- Q_ CO ι } CO ** T3 £ « m O o co > C CL co : LU o

---------------------έ>° o

Ajisuami %

Ajisuaiui %

LU

O

........—Aiisueiui %

E

Aijsueiui %

LO c +-> LO t—I I c o LO 1 > > CO > c > > CO 00 _I 00 I 1 c 00 _1 LU L_ LU ^“1 LO LU L_ o t—I LO I LO + o t—I t—1 LO 1 1 CO _1 o t—1 to 1 LO + CJ LO s_ U =E ΞΕ =E

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FIG. 15

50 pg/ml

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Virus ID

10E8 Clade Varl

10E8 10E8

Var4 Var5

FIG. 16A

10E8 Virus ID

10E8 10E8 10E8

Clade Varl Var4 Var5 10E8

0260.v5.c36 A 9.170 11.500 10.400 9.870 M02138 AE 0330.v4.c3 A 1.510 2.090 1.740 1.120 R1166.C1 AE 0.415 0.431 0.534 0.488 0439.v5.cl A 0.982 1.490 1.490 1.230 R2184.C4 AE 0.662 0.594 0.518 0.576 3365.v2.c20 A 1.310 1.240 1.280 1.600 R3265.C6 AE 1.540 1.990 1.480 1.580 3415.vl.cl A 3.520 4.400 4.240 4.690 TH023.6 AE 3718.v3.cll A 1.130 1.840 1.680 0.838 TH966.8 AE 0.034 0.046 0.051 0.039 398-F1F620 A 1.080 1.470 0.857 0.704 TH976.17 AE 0.582 0.473 0.359 0.392 BB201.B42 A 0.680 0.849 1.010 0.613 235-47 AG 0.119 0.121 0.112 0.244 BB539.2B13 A 242-14 AG 0.460 0.431 0.431 0.568 BG505.W6M.C2 A 0.469 0.555 0.803 0.689 263-8 AG 0.103 0.104 0.192 0.229 BI369.9A A 0.315 0.393 0.414 0.356 269-12 AG 0.025 0.095 0.142 0.124 BS208.B1 A 0.252 0.324 0.332 0.319 271-11 AG 0.686 0.882 0.836 0.891 KER2008.12 A 100 100 100 100 928-28 AG 0.013 0.018 0.021 0.079 KER2018.il A 1.600 2.320 2.250 1.890 DJ263.8 AG 0.0008 0.002 0.003 0.009 KNH1209.18 A 0.315 0.468 0.325 0.406 T250-4 AG 0.745 0.823 0.804 1.070 MB201.A1 A 0.346 0.492 0.374 0.411 T251-18 AG 0.426 0.336 0.307 0.666 MB539.2B7 A 28.700 30.300 21.500 100.000 T253-11 AG 0.960 1.160 0.986 1.210 MI369.A5 A 0.835 1.170 0.904 0.671 T255-34 AG 0.149 0.183 0.202 0.228 MS208.A1 A 0.546 0.889 0.392 0.187 T257-31 AG 0.435 0.304 0.368 0.336 Q23.17 A 0.931 1.180 0.667 0.461 T266-60 AG 100 100 100 100 Q259.17 A 4.100 3.370 5.060 4.760 T278-50 AG 0.303 0.293 0.253 0.357 Q769.d22 A 1.390 1.590 1.720 1.910 T280-5 AG Q769.h5 A 2.530 2.840 3.000 2.890 T33-7 AG 0.897 1.010 1.240 0.818 Q842.dl2 A 2.320 1.990 2.600 2.820 3988.25 B 0.029 0.050 0.080 0.070 QH209.14M.A2 A 1.080 0.867 0.887 1.300 5768.04 B 1.860 2.190 2.540 1.630 RW020.2 A 1.020 1.020 0.883 0.902 6101.10 B 0.001 0.002 0.002 0.001 UG037.8 A 0.035 0.080 0.058 0.048 6535.3 B 0.026 0.046 0.078 0.190 246-F3.C10.2 AC 0.072 0.088 0.128 0.210 7165.18 B 0.232 0.258 0.421 0.659 3301.V1.C24 AC 3.100 3.310 3.210 2.970 45 01dG5 B 0.198 0.182 0.276 0.106 3589.V1.C4 AC 3.370 5.150 4.860 5.770 89.6.DG B 0.264 0.126 0.378 0.318 6540.v4.cl AC 2.510 2.320 2.870 2.240 AC10.29 B 0.016 0.046 0.050 0.102 6545.V4.C1 AC 1.930 1.740 3.200 2.540 ADA.DG B 0.010 0.023 0.036 0.055 0815.V3.C3 ACD 1.080 1.040 0.788 0.491 Bal.Ol B 0.412 0.531 0.661 0.421 6095.V1.C10 ACD 0.0003 0.0003 0.0003 0.0005 BaL.26 B 0.506 0.524 0.811 0.518 3468.V1.C12 AD 0.318 0.307 0.291 0.381 BG1168.01 B 0.142 0.305 0.233 0.396 Q168.a2 AD 0.709 0.964 0.466 0.463 BL01.DG B 0.427 0.505 0.521 0.362 Q461.e2 AD 2.080 2.270 2.140 2.290 BR07.DG B 0.064 0.099 0.162 0.118 620345.cl AE 0.620 0.462 0.761 0.989 BX08.16 B 0.089 0.108 0.184 0.213 BJOX009000.02.4 AE 0.196 0.229 0.286 0.251 CAAN.A2 B 2.350 2.330 3.570 1.450 BJOX010000.06.2 AE 0.014 0.049 0.055 0.060 CNE10 B 0.012 0.005 0.019 0.014 BJOX025000.01.1 AE 0.095 0.234 0.209 0.228 CNE12 B 0.119 0.130 0.240 0.301 BJOX028000.10.3 AE 0.021 0.125 0.110 0.167 CNE14 B 0.437 0.309 0.508 0.151 C1080.C3 AE 0.030 0.038 0.112 0.108 CNE4 B 0.062 0.057 0.121 0.059 C2101.cl AE 1.160 1.120 1.300 1.200 CNE57 B 0.065 0.065 0.068 0.059 C3347.cH AE 0.002 0.005 0.002 0.019 HO86.8 B 0.373 0.275 0.368 0.326 C4118.09 AE 0.319 0.268 0.384 0.421 HT593.1 B 0.029 0.031 0.038 0.049 CM244.ecl AE 0.714 0.890 0.432 0.365 HXB2.DG B 0.0003 .0003 .0003 0.003 CNE3 AE 0.924 0.528 1.280 1.370 JRCSF.JB B 0.636 0.632 0.876 0.429 CNE5 AE 0.792 0.678 0.900 1.170 JRFL.JB B 0.219 0.313 0.318 0.174 CNE55 AE 0.026 0.027 0.026 0.038 MN.3 B .0003 .0003 .0003 .0003 CNE56 AE 0.022 0.029 0.026 0.060 PVO.04 B 1.670 2.650 1.540 1.600 CNE59 AE 0.0003 .00071 0.0003 0.001 QH0515.01 B 1.890 2.450 2.650 2.250 CNE8 AE 0.051 0.096 0.090 0.140

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FIG 10E8 10E8 10E8 Virus ID Clade Varl Var4 Var5 10E8 16B 10E8 10E8 10E8 Virus ID Clade Varl Var4 Var5 10E8 QH0692.42 B 0.474 0.492 0.348 0.531 CAP45.G3 C 0.531 0.787 0.694 0.722 REJO.67 B 0.419 0.451 0.425 0.302 Cell76.A3 C 0.226 0.311 0.341 0.252 RHPA.7 B 0.925 0.981 0.968 1.010 CE703010217.B6 C 0.049 0.137 0.113 0.096 SC422.8 B 0.356 0.343 0.363 0.343 CNE30 C 0.370 0.354 0.327 0.456 SF162.LS B 0.639 0.701 0.673 0.245 CNE31 C 0.805 0.785 1.220 1.320 SS1196.01 B 0.165 0.119 0.173 0.244 CNE53 C 0.108 0.130 0.091 0.213 THR0.18 B 0.074 0.108 0.105 0.092 CNE58 C 0.119 0.193 0.240 0.229 TRJO.58 B 2.980 3.130 2.390 1.130 DU 123.06 C 0.053 0.046 0.062 0.132 TRO.ll B 0.059 0.072 0.048 0.028 DU 151.02 C 0.481 0.321 0.499 0.461 WITO.33 B 0.051 0.045 0.042 0.031 DU 156.12 C 0.004 0.003 0.008 0.023 X2278.C2.B1 B 0.605 0.611 0.671 0.442 DU172.17 C 0.018 0.011 0.030 0.057 YU2.DG B 2.070 1.990 2.200 1.170 DU422.01 C 0.247 0.202 0.337 0.224 BJOX002000.03.2 BC 0.147 0.254 0.502 0.384 MW965.26 C 0.000 0.000 0.000 0.001 CH038.12 BC 0.230 0.265 0.275 0.271 SO18.18 C 1.080 0.675 1.150 1.600 CH070.1 BC 5.500 5.730 7.840 6.650 TV1.29 C 0.408 0.421 0.310 0.248 CH117.4 BC 0.225 0.222 0.303 0.270 TZA125.17 C 0.250 0.218 0.154 0.217 CH119.10 BC 0.664 0.344 0.667 0.591 TZBD.02 C 2.010 1.960 1.720 1.410 CH181.12 BC 0.683 0.602 1.000 0.754 ZA012.29 c 1.290 1.660 1.670 1.470 CNE15 BC 0.503 0.568 0.742 0.844 ZM 106.9 c 100 100 100 100 CNE19 BC 0.146 0.155 0.154 0.251 ZM 109.4 c 0.063 0.118 0.096 0.161 CNE20 BC 0.066 0.102 0.132 0.131 ZM 135.10a c 0.020 0.040 0.051 0.033 CNE21 BC 0.509 0.354 0.499 0.979 ZM 176.66 c 0.386 0.590 0.661 0.267 CNE40 BC 0.000 0.001 0.001 0.001 ZM 197.7 c 0.015 0.022 0.040 0.055 CNE7 BC 0.072 0.093 0.093 0.130 ZM214.15 c 2.040 2.140 2.920 2.220 286.36 C 0.529 0.746 0.853 1.190 ZM215.8 c 0.008 0.026 0.024 0.044 288.38 C 0.385 0.491 0.462 0.435 ZM233.6 c 0.122 0.214 0.194 0.270 0013095-2.11 C 0.002 0.002 0.002 0.009 ZM249.1 c 0.704 0.682 0.752 0.830 001428-2.42 C 2.240 0.726 1.890 1.710 ZM53.12 c 2.300 2.160 3.080 2.620 0077 V1.C16 C 1.410 1.310 2.600 1.860 ZM55.28a c 2.750 2.560 2.980 2.340 00836-2.5 C 0.538 0.445 0.452 0.666 3326.V4.C3 CD 1.180 1.260 0.664 1.400 0921.V2.C14 C 1.160 1.680 1.130 0.908 3337.V2.C6 CD 0.883 1.400 0.825 1.090 16055-2.3 C 1.040 1.480 1.280 1.100 3817.v2.c59 CD 0.543 1.210 0.679 0.229 16845-2.22 C 0.007 0.010 0.010 0.020 191821.E6.1 D 16936-2.21 C 0.205 0.353 0.259 0.264 231965.cl D 8.010 8.660 9.400 11.000 25710-2.43 C 0.011 0.025 0.053 0.064 247-23 D 0.245 0.441 0.403 0.344 25711-2.4 C 0.652 0.595 0.669 0.516 3016.v5.c45 D 0.610 0.604 0.580 0.710 25925-2.22 C 0.234 0.461 0.456 0.402 57128.vrcl5 D 0.123 0.067 0.195 0.212 26191-2.48 C 1.550 0.969 1.260 1.830 6405.v4.c34 D 0.781 0.617 1.080 0.461 3168.V4.C10 C 2.020 2.940 1.920 2.830 A03349Ml.vrc4a D 0.169 0.332 0.288 0.270 3637.V5.C3 C 1.530 2.480 2.120 2.120 A07412Ml.vrcl2 D 0.094 0.119 0.145 0.140 3873.V1.C24 C 3.380 3.300 4.700 5.510 NKU3006.ecl D 0.726 1.020 0.616 0.673 6322.V4.C1 C 0.372 0.691 0.706 0.923 UG021.16 D 6471.V1.C16 C 4.650 5.370 6.740 4.980 UG024.2 D 6631.V3.C10 C 1.360 1.440 2.300 0.934 P0402.c2.ll G 0.032 0.055 0.081 0.057 6644.V2.C33 C 0.008 0.011 0.029 0.013 P1981.C5.3 G 0.004 0.010 0.005 0.024 6785.V5.C14 C 0.756 1.100 0.924 0.701 XI193.cl G 0.222 0.201 0.354 0.341 6838.V1.C35 c 0.168 0.153 0.354 0.292 X1254.C3 G 2.070 2.370 2.940 3.670 96ZM651.02 c 0.004 0.009 0.010 0.033 X1632.S2.B10 G 0.289 0.285 0.415 0.387 BR025.9 c 0.235 0.498 0.398 0.307 X2088.C9 G 100 100 100 100 CAP210.E8 c 0.346 0.439 0.321 0.474 X2131.C1.B5 G 0.003 0.010 0.015 0.039 CAP244.D3 c 0.239 0.334 0.307 0.369 SIVmac251.30.SG3 NA >50 >50 >50 >50 CAP256.206.C9 c 0.657 0.913 0.804 0.713 SVA.MLV NA >50 >50 >50 >50

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FIG. 16C

10E8 Varl 10E8 Var4 10E8 Var5 10E8 # Viruses 200 200 200 200 Total Virus Neutralized IC50 <50ug/ml 196 196 196 195 IC50 <10ug/ml 195 194 194 194 IC50 <1.0ug/ml 148 143 146 145 IC50 <0.1ug/ml 53 46 42 38 IC50 <0.01ug/ml 17 13 12 9 % Virus Neutralized IC50 <50ug/ml 98 98 98 98 IC50 <10ug/ml 98 97 97 97 IC50 <1.0ug/ml 74 72 73 73 IC50 <0.1ug/ml 27 23 21 19 IC50 <0.01ug/ml 9 7 6 5 Median IC50 0.397 0.435 0.418 0.392 Geometric Mean 0.228 0.276 0.307 0.315

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FIG.

10E8

Virus ID

10E8 10E8

Clade Varl Var4

10E8

Var5

0260.v5.c36 A 14.100 30.200 26.700 21.700 0330.v4.c3 A 4.150 6.580 4.720 3.640 0439.v5.cl A 3.420 5.210 4.170 3.950 3365.v2.c20 A 4.150 4.980 3.940 4.560 3415.vl.cl A 9.390 11.700 14.000 11.500 3718.v3.cll A 4.360 6.970 6.840 4.420 398-F1 F6 20 A 5.440 10.600 7.230 6.170 BB201.B42 A 3.490 4.240 3.620 1.960 BB539.2B13 A BG505.W6M.C2 A 1.940 2.690 2.390 2.140 BI369.9A A 1.340 2.000 1.570 1.290 BS208.B1 A 2.130 3.380 2.170 3.270 KER2008.12 A 100 100 100 100 KER2018.il A 5.670 8.460 6.460 7.160 KNH1209.18 A 2.030 2.700 1.900 2.390 MB201.A1 A 1.800 2.270 1.730 1.360 MB539.2B7 A 100 100 100 100 MI369.A5 A 2.360 3.630 2.770 1.770 MS208.A1 A 1.940 3.890 1.530 1.140 Q23.17 A 2.660 4.280 3.050 1.600 Q259.17 A 9.750 11.600 11.600 12.000 Q769.d22 A 3.570 5.220 3.990 4.470 Q769.h5 A 6.020 9.460 8.650 7.440 Q842.dl2 A 5.530 7.200 7.020 7.580 QH209.14M.A2 A 3.880 4.940 3.590 4.090 RW020.2 A 2.810 3.820 3.820 2.920 UG037.8 A 0.497 0.677 0.356 0.353 246-F3.C10.2 AC 1.200 1.780 1.130 1.490 3301.V1.C24 AC 8.590 11.200 9.950 9.500 3589.V1.C4 AC 7.560 13.300 10.900 11.700 6540.v4.cl AC 8.050 9.240 9.630 7.010 6545.V4.C1 AC 7.570 7.590 8.650 7.500 0815.V3.C3 ACD 3.310 3.970 2.870 1.810 6095.V1.C10 ACD 0.0007 0.001 0.001 0.004 3468.V1.C12 AD 1.610 2.390 1.650 2.040 Q168.a2 AD 2.590 4.620 2.930 2.880 Q461.e2 AD 4.750 5.750 4.880 4.680 620345.cl AE 3.610 4.180 3.540 3.730 BJOX009000.02.4 AE 1.580 2.240 1.670 1.470 BJOX010000.06.2 AE 0.325 0.524 0.572 0.476 BJOX025000.01.1 AE 1.540 2.200 1.630 1.540 BJOX028000.10.3 AE 0.433 1.140 0.864 0.876 C1080.C3 AE 0.447 0.582 0.831 0.613 C2101.cl AE 4.010 5.260 4.970 4.120 C3347.cH AE 0.019 0.053 0.050 0.089 C4118.09 AE 2.760 3.360 2.900 2.300 CM244.ecl AE 3.100 3.870 2.930 1.460 CNE3 AE 3.530 2.920 4.640 4.010 CNE5 AE 2.330 4.130 3.590 2.520 CNE55 AE 0.566 0.594 0.558 0.605 CNE56 AE 0.236 0.489 0.205 0.314 CNE59 AE 0.002 0.004 0.003 0.010

^z 10E8 10E8 10E8

Virus ID Clade Varl Var4 Var5 10E8

M02138 AE R1166.C1 AE 2.000 2.600 1.870 2.020 R2184.C4 AE 2.190 2.700 2.050 2.200 R3265.C6 AE 8.400 9.750 8.610 9.280 TH023.6 AE TH966.8 AE 0.392 0.440 0.339 0.291 TH976.17 AE 2.390 2.340 1.850 1.750 235-47 AG 0.664 1.110 0.596 0.786 242-14 AG 1.870 2.170 2.060 3.170 263-8 AG 0.864 0.974 0.909 0.991 269-12 AG 0.305 0.793 0.479 0.475 271-11 AG 3.890 4.820 3.830 4.340 928-28 AG 0.243 0.639 0.276 0.365 DJ263.8 AG 0.050 0.054 0.070 0.100 T250-4 AG 2.720 3.090 3.460 3.450 T251-18 AG 2.230 2.860 2.180 2.550 T253-11 AG 3.100 4.590 4.120 4.050 T255-34 AG 1.220 1.810 1.470 1.140 T257-31 AG 1.740 2.150 1.840 1.580 T266-60 AG 100 100 100 100 T278-50 AG 1.640 1.970 1.600 2.100 T280-5 AG T33-7 AG 3.440 2.730 4.410 2.830 3988.25 B 0.376 0.607 0.409 0.293 5768.04 B 5.870 9.010 8.640 5.260 6101.10 B 0.020 0.025 0.019 0.005 6535.3 B 0.567 1.150 1.120 1.280 7165.18 B 1.540 2.230 2.290 2.710 45 01dG5 B 1.360 1.310 1.590 0.703 89.6.DG B 1.320 1.340 1.500 1.480 AC10.29 B 0.372 0.577 0.511 0.512 ADA.DG B 0.229 0.342 0.363 0.358 Bal.Ol B 2.310 3.410 2.850 1.910 BaL.26 B 2.880 3.180 3.890 2.390 BG1168.01 B 0.806 1.590 1.040 1.480 BL01.DG B 2.900 3.230 2.600 1.570 BR07.DG B 0.539 0.781 0.891 0.445 BX08.16 B 1.050 1.660 1.310 1.300 CAAN.A2 B 8.050 12.000 10.900 5.700 CNE10 B 0.335 0.304 0.331 0.169 CNE12 B 0.789 0.939 1.040 1.090 CNE14 B 2.030 1.910 2.560 0.649 CNE4 B 0.824 0.918 1.170 0.437 CNE57 B 0.510 0.743 0.538 0.317 HO86.8 B 2.640 2.210 2.810 1.520 HT593.1 B 0.404 0.497 0.421 0.285 HXB2.DG B 0.001 0.004 0.003 0.015 JRCSF.JB B 3.080 3.750 3.710 1.890 JRFL.JB B 1.330 1.530 1.410 0.768 MN.3 B 0.0007 0.001 0.0009 0.001 PVO.04 B 7.080 10.100 7.470 6.430 QH0515.01 B 5.100 8.390 7.070 5.540

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10E8 10E8 10E8 FIG. 16E 10E8 10E8 10E8 Virus ID Clade Varl Var4 Var5 10E8 Virus ID Clade Varl Var4 Var5 10E8

QH0692.42 B 2.750 4.090 2.850 2.350 CAP45.G3 C 3.250 3.720 2.770 3.410 REJO.67 B 2.020 2.440 1.990 1.180 Cell76.A3 C 1.180 1.720 1.420 1.150 RHPA.7 B 5.660 8.010 5.520 5.100 CE703010217.B6 C 0.862 1.190 0.888 0.679 SC422.8 B 1.310 1.570 1.600 1.150 CNE30 C 2.190 1.960 2.080 2.290 SF162.LS B 2.790 3.880 2.960 1.060 CNE31 C 2.630 3.120 3.160 3.570 SS1196.01 B 1.230 1.250 1.050 1.250 CNE53 C 0.747 0.910 0.755 1.010 THR0.18 B 0.684 0.774 0.587 0.587 CNE58 C 0.997 1.800 1.220 1.090 TRJO.58 B 7.740 9.560 8.350 4.180 DU123.06 C 0.421 0.497 0.474 0.423 TRO.ll B 0.625 0.717 0.358 0.286 DU151.02 C 1.830 1.760 1.970 1.710 WITO.33 B 0.519 0.445 0.395 0.305 DU156.12 C 0.059 0.053 0.102 0.120 X2278.C2.B1 B 3.610 3.360 3.480 2.240 DU172.17 C 0.220 0.203 0.281 0.238 YU2.DG B 8.260 10.400 10.100 5.460 DU422.01 C 1.260 1.430 1.430 0.812 BJOX002000.03.2 BC 1.000 1.880 1.800 1.560 MW965.26 C 0.001 0.002 0.002 0.007 CH038.12 BC 1.900 2.170 1.770 1.410 SO18.18 C 3.390 3.890 3.970 4.480 CH070.1 BC 15.90 19.80 14.70 13.50 TV1.29 C 1.150 1.320 0.826 0.719 CH117.4 BC 1.150 0.791 1.260 0.859 TZA125.17 C 1.700 1.770 1.360 1.190 CH119.10 BC 2.710 2.490 3.320 2.360 TZBD.02 C 4.450 6.410 5.300 4.310 CH181.12 BC 2.560 3.710 3.300 2.790 ZA012.29 c 3.870 5.270 4.770 4.120 CNE15 BC 2.580 3.120 2.940 2.970 ZM106.9 c 100 100 100 100 CNE19 BC 1.190 1.100 1.060 1.110 ZM109.4 c 1.090 1.420 1.150 1.070 CNE20 BC 0.765 0.768 0.955 0.732 ZM135.10a c 0.356 0.575 0.535 0.408 CNE21 BC 3.150 2.740 3.390 3.250 ZM176.66 c 2.300 3.230 2.700 1.730 CNE40 BC .0007 0.011 0.008 0.009 ZM197.7 c 0.190 0.365 0.293 0.369 CNE7 BC 0.577 0.786 0.645 0.603 ZM214.15 c 5.370 8.230 7.960 5.980 286.36 C 3.540 4.630 4.810 5.000 ZM215.8 c 0.106 0.247 0.249 0.230 288.38 C 2.470 3.940 2.340 3.080 ZM233.6 c 0.708 0.974 0.873 0.737 0013095-2.11 C 0.025 0.015 0.046 0.077 ZM249.1 c 2.150 2.940 2.740 2.270 001428-2.42 C 6.200 5.860 6.960 6.280 ZM53.12 c 5.750 7.730 7.840 6.720 0077 V1.C16 C 6.740 8.460 8.670 7.110 ZM55.28a c 6.560 8.330 7.730 6.780 00836-2.5 C 1.520 1.910 2.020 1.770 3326.V4.C3 CD 4.200 5.560 3.250 4.290 0921.V2.C14 C 3.530 5.010 3.860 3.030 3337.V2.C6 CD 4.100 6.510 3.680 4.870 16055-2.3 C 3.560 5.270 4.060 3.310 3817.v2.c59 CD 2.170 4.480 2.650 1.430 16845-2.22 C 0.085 0.161 0.200 0.172 191821.E6.1 D 16936-2.21 C 1.290 2.000 1.480 1.310 231965.cl D 17.500 24.30 18.70 20.400 25710-2.43 C 0.104 0.297 0.301 0.304 247-23 D 1.060 2.010 1.260 1.290 25711-2.4 C 1.880 2.000 2.280 1.690 3016.v5.c45 D 2.240 2.530 2.830 2.170 25925-2.22 C 1.470 2.510 1.900 1.530 57128.vrcl5 D 1.450 1.750 1.420 1.500 26191-2.48 C 4.500 4.700 5.550 4.900 6405.v4.c34 D 4.170 5.330 5.160 1.800 3168.V4.C10 C 5.050 10.500 7.230 8.180 A03349Ml.vrc4a D 0.727 1.380 1.040 0.663 3637.V5.C3 C 4.990 7.650 5.600 6.680 A07412Ml.vrcl2 D 0.989 1.090 1.110 0.873 3873.V1.C24 C 10.80 0 12.800 14.300 15.700 NKU3006.ecl D 2.870 3.890 2.370 2.460 6322.V4.C1 C 2.670 4.270 3.050 3.680 UG021.16 D 6471.V1.C16 C 13.30 14.60 16.30 14.90 UG024.2 D 6631.V3.C10 C 4.610 5.660 7.090 3.360 P0402.c2.ll G 0.525 0.645 0.291 0.460 6644.V2.C33 C 0.142 0.229 0.277 0.124 P1981.C5.3 G 0.066 0.152 0.072 0.124 6785.V5.C14 C 2.990 4.230 3.840 2.420 X1193.C1 G 0.952 1.210 1.500 1.150 6838.V1.C35 c 1.010 1.460 1.340 1.010 X1254.C3 G 7.200 8.500 8.500 15.700 96ZM651.02 c 0.070 0.130 0.300 0.177 X1632.S2.B10 G 1.720 2.230 1.880 1.760 BR025.9 c 1.330 2.390 1.570 1.110 X2088.C9 G 100 100 100 100 CAP210.E8 c 1.920 2.950 2.040 2.010 X2131.C1.B5 G 0.071 0.170 0.139 0.175 CAP244.D3 c 1.390 2.370 1.710 1.480 SIVmac251.30.SG 3 NA >50 >50 >50 >50 CAP256.206.C9 c 3.190 4.480 3.940 2.970 SVA.MLV NA >50 >50 >50 >50

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FIG. 16F

10E8 Varl 10E8 Var4 10E8 Var5 10E8 # Viruses 200 200 200 200 Total Virus Neutralized IC80 <50ug/ml 195 195 195 195 IC80 <10ug/ml 190 181 185 186 IC80 <1.0ug/ml 57 49 51 57 IC80 <0.1ug/ml 15 11 11 9 IC80 <0.01ug/ml 6 5 6 5 % Virus Neutralized IC80 <50ug/ml 98 98 98 98 IC80 <10ug/ml 95 91 93 93 IC80 <1.0ug/ml 29 25 26 29 IC80 <0.1ug/ml 8 6 6 5 IC80 <0.01ug/ml 3 3 3 3 Median IC80 1.940 2.390 2.040 1.710 Geometric Mean 1.271 1.753 1.532 1.370

Days post administration

40E8 {A13V1O0} 10E8V4(A13V188) 10E8v4LS (A13V033) -S0E8V4LS (A13V076)

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FIG. 18B

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FIG. 18D

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FIG. 22

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FIG. 23A

IC50 clade virus

A KER2008.12.SG3 A RW020.2.SG3 ACD 6095.V1.C10 AE CM244.ecl.SG3 AE TH966.8 B 6101.1 B CAAN.A2.SG3 B PVO.04.SG3 B YU2.DG.SG3 C CNE31.SG3 C ZM106.9.SG3 C ZM215.8 C ZM55.28a.SG3 Median IC50 Geometric Mean Fold Improvement over10E8v4 V5R

SlOOcF

10E8- 1053 10E8- 1054 10E8- 1055 2.4600 0.8380 3.4700 0.0530 0.0580 0.1440 0.0006 0.0006 0.0003 0.0080 0.0020 0.0240 0.0006 0.0006 0.0020 0.0006 0.0006 0.0020 0.2250 0.2170 0.3430 0.1660 0.1540 0.3220 0.0860 0.1080 0.1350 0.0340 0.0300 0.0790 50.0000 12.3000 25.0000 0.0006 0.0006 0.0050 0.1000 0.0880 0.1150 0.053 0.058 0.115 0.034 0.025 0.063 1.8 2.4 1.0

10E8- 1056 10E8- 1057 10E8- 1058 0.7980 0.7690 0.7020 0.0500 0.1200 0.0460 0.0006 0.0003 0.0003 0.0050 0.0140 0.0120 0.0006 0.0030 0.0003 0.0006 0.0020 0.0006 0.1390 0.1880 0.0870 0.1460 0.4990 0.1560 0.0750 0.1930 0.0580 0.0490 0.0780 0.0370 50.0000 25.0000 9.1400 0.0006 0.0060 0.0020 0.0850 0.1600 0.0550 0.050 0.120 0.046 0.029 0.058 0.023 2.1 1.1 2.6

10E8- 1059 10E8- 1060 10E8- 1061 50.0000 50.0000 50.0000 0.0350 0.0390 0.0230 0.0006 0.0006 0.0006 0.0180 0.0100 0.0100 0.0006 0.0006 0.0006 0.0006 0.0006 0.0006 0.2430 0.1360 0.1010 0.0700 0.1190 0.0570 0.0370 0.0540 0.0130 0.0350 0.0360 0.0170 50.0000 50.0000 50.0000 0.0006 0.0006 0.0006 0.0650 0.0510 0.0350 0.035 0.039 0.017 0.038 0.036 0.027 1.6 1.7 2.3

IC50 10E8- 1062 10E8- 1063 10E8- 1064 10E8- 1022 10e8 clade virus A KER2008.12.SG3 1.3400 1.0400 1.5800 42.0000 50.0000 A RW020.2.SG3 0.0340 0.0430 0.0320 0.0850 1.3500 ACD 6095.V1.C10 0.0006 0.0003 0.0006 0.0006 0.0006 AE CM244.ecl.SG3 0.0080 0.0180 0.0120 0.0480 0.2160 AE TH966.8 0.0006 0.0003 0.0006 0.0006 0.0400 B 6101.1 0.0006 0.0030 0.0006 0.0006 0.0030 B CAAN.A2.SG3 0.0930 0.1520 0.2070 0.1890 2.8700 B PVO.04.SG3 0.1580 0.2010 0.0800 0.2550 2.5400 B YU2.DG.SG3 0.0840 0.1340 0.0470 0.0900 1.1200 C CNE31.SG3 0.0300 0.0440 0.0200 0.0620 1.1800 C ZM106.9.SG3 42.3000 25.0000 19.9000 50.0000 50.0000 C ZM215.8 0.0006 0.0020 0.0007 0.0020 0.0630 C ZM55.28a.SG3 0.0500 0.0760 0.0420 0.1580 2.4400 Median IC50 0.034 0.044 0.032 0.085 1.180 Geometric Mean 0.027 0.036 0.025 0.061 0.483 Fold Improvement 2.3 1.7 2.5 1.0

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FIG. 23B

IC80 10E8- 1053 10E8- 1054 10E8- 1055 10E8- 1056 10E8- 1057 10E8- 1058 10E8- 1059 10E8- 1060 10E8- 1061 clade virus A KER2008.12.SG3 50.0000 12.4000 25.0000 7.9200 5.5000 6.2700 50.0000 50.0000 50.0000 A RWO2O.2.SG3 0.2310 0.2380 0.4830 0.2370 0.3780 0.1570 0.1570 0.1620 0.1040 ACD 6095.V1.C10 0.0006 0.0006 0.0003 0.0006 0.0003 0.0003 0.0006 0.0006 0.0006 AE CM244.ecl.SG3 0.1360 0.1050 0.1890 0.0770 0.1210 0.0660 0.1180 0.0770 0.0640 AE TH966.8 0.0080 0.0110 0.0240 0.0120 0.0250 0.0100 0.0006 0.0030 0.0006 B 6101.1 0.0050 0.0110 0.0200 0.0140 0.0230 0.0070 0.0080 0.0110 0.0070 B CAAN.A2.SG3 0.7410 0.7970 1.1000 0.6120 0.6790 0.4210 1.3100 0.7040 0.6440 B PVO.04.SG3 0.8430 0.9000 1.2400 0.7100 1.4100 0.7850 0.5680 0.6730 0.2870 B YU2.DG.SG3 0.6240 0.6450 0.8160 0.5340 0.9670 0.4820 0.4700 0.5390 0.2140 C CNE31.SG3 0.1630 0.1520 0.3010 0.1740 0.2600 0.1280 0.1550 0.1380 0.0760 C ZM106.9.SG3 50.0000 50.0000 25.0000 50.0000 25.0000 25.0000 50.0000 50.0000 50.0000 C ZM215.8 0.0120 0.0210 0.0280 0.0170 0.0320 0.0190 0.0110 0.0140 0.0070 C ZM55.28a.SG3 0.3500 0.3310 0.4390 0.3250 0.5190 0.2680 0.3090 0.2560 0.1630 Median IC80 0.231 0.238 0.439 0.237 0.378 0.157 0.157 0.162 0.104 Geometric Mean Fold 0.193 0.194 0.266 0.177 0.227 0.132 0.153 0.167 0.103 improvement 1.5 1.5 1.1 1.6 1.2 2.2 1.9 1.7 2.8

IC80 10E8- 1062 10E8- 1063 10E8- 1064 10E8- 1022 10e8 clade virus A KER2008.12.SG3 30.7000 7.7600 14.4000 50.0000 50.0000 A RW020.2.SG3 0.1600 0.1830 0.1360 0.3910 4.5700 ACD 6095.V1.C10 0.0006 0.0003 0.0006 0.0006 0.0050 AE CM244.ecl.SG3 0.0930 0.0970 0.0640 0.3150 1.8400 AE TH966.8 0.0080 0.0100 0.0050 0.0090 0.1580 B 6101.1 0.0130 0.0270 0.0090 0.0140 0.0630 B CAAN.A2.SG3 0.6320 0.5810 0.7450 0.9630 9.5400 B PVO.04.SG3 0.7430 0.7440 0.4830 1.1200 11.6000 B YU2.DG.SG3 0.6380 0.7390 0.4000 0.8650 8.0100 C CNE31.SG3 0.1240 0.1720 0.1040 0.3080 4.7000 C ZM106.9.SG3 50.0000 25.0000 50.0000 50.0000 50.0000 C ZM215.8 0.0110 0.0120 0.0060 0.0170 0.5800 C ZM55.28a.SG3 0.2670 0.2960 0.2030 0.6630 7.3300 Median IC80 0.160 0.183 0.136 0.391 4.700 Geometric Mean 0.176 0.162 0.130 0.283 2.130 Fold improvement 1.6 1.7 2.2 1

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FIG. 23C

clade IC50 virus 10E8- 1065 10E8- 1068 10E8- 1069 10E8- 1070 10E8- 1072 10E8- 1073 10E8- 1074 10E8-1075 10E8- 1076 A KER2008.12.SG3 1.5500 2.9900 3.6200 3.6900 5.8600 0.9370 4.6400 5.7700 1.9000 A RWO2O.2.SG3 0.0550 0.0970 0.0580 0.1490 0.0850 0.0520 0.0660 0.0700 0.0900 ACD 6095.V1.C10 0.0004 0.0006 0.0003 0.0006 <0.0003 0.0004 0.0003 0.0004 0.0005 AE CM244.ecl.SG3 0.0260 0.0290 0.0240 0.0520 0.0330 0.0320 0.0350 0.0410 0.0540 AE TH966.8 0.0050 0.0080 0.0050 0.0110 0.0060 0.0050 0.0060 0.0060 0.0080 B 6101.1 <0.0003 0.0020 0.0010 0.0040 0.0010 0.0010 0.0020 0.0020 0.0020 B CAAN.A2.SG3 0.0940 0.1220 0.1030 0.1470 0.1410 0.0610 0.1260 0.1010 0.1020 B PVO.04.SG3 0.0890 0.1350 0.0700 0.2570 0.0880 0.1290 0.1730 0.1440 0.2260 B YU2.DG.SG3 0.0690 0.0790 0.0350 0.1080 0.0400 0.0340 0.0430 0.0510 0.0720 C CNE31.SG3 0.0490 0.0750 0.0360 0.0750 0.0520 0.0330 0.0410 0.0370 0.0530 C ZM106.9.SG3 >25 >25 >20 18.9000 >25 16.2000 18.1000 >25 13.2000 C ZM215.8 0.0060 0.0060 0.0030 0.0070 0.0050 0.0050 0.0040 0.0050 0.0060 C ZM55.28a.SG3 0.0890 0.1070 0.0670 0.1330 0.1150 0.0680 0.0910 0.0840 0.1410 Median IC50 0.055 0.077 0.036 0.108 0.052 0.034 0.043 0.046 0.072 Geometric Mean 0.034 0.036 0.022 0.078 0.045 0.036 0.049 0.031 0.057

clade IC50 virus 10E8- 1077 10E8- 1078 10E8- 1079 10E8- 1080 10E8- 1081 10E8- 1082 10E8- 1083 10E8-1084 10E8- 1086 A KER2008.12.SG3 4.0300 3.8500 2.4000 >25 >25 >25 >25 >25 6.5600 A RW020.2.SG3 0.1000 0.1010 0.0800 0.1080 0.0420 0.0880 0.4050 0.0850 0.0630 ACD 6095.V1.C10 0.0004 0.0005 0.0004 0.0006 <0.0003 0.0004 0.0030 <0.0003 <0.0003 AE CM244.ecl.SG3 0.0450 0.0690 0.0530 0.0430 0.0350 0.0360 0.2640 0.0880 0.0500 AE TH966.8 0.0110 0.0080 0.0070 0.0060 0.0020 0.0050 0.0280 0.0040 0.0030 B 6101.1 0.0020 0.0020 0.0010 0.0030 0.0020 0.0050 0.1280 <0.0003 <0.0003 B CAAN.A2.SG3 0.1100 0.1540 0.1480 0.1550 0.1310 0.2240 0.6820 0.1830 0.0940 B PVO.04.SG3 0.2170 0.2160 0.2250 0.1540 0.0490 0.1310 1.3300 0.1060 0.0800 B YU2.DG.SG3 0.0640 0.0490 0.0480 0.0830 0.0140 0.0700 0.2450 0.0600 0.0390 C CNE31.SG3 0.0510 0.0510 0.0500 0.0540 0.0190 0.0360 0.2640 0.0500 0.0390 C ZM106.9.SG3 >25 >25 >25 >25 >25 >25 >25 >25 >25 C ZM215.8 0.0070 0.0040 0.0050 0.0050 0.0020 0.0040 0.0330 0.0050 0.0040 C ZM55.28a.SG3 0.1280 0.1270 0.1230 0.1010 0.0340 0.0880 0.3640 0.0890 0.0780 Median IC50 0.058 0.060 0.052 0.054 0.027 0.036 0.264 0.085 0.057 Geometric Mean 0.039 0.038 0.033 0.025 0.015 0.023 0.149 0.045 0.054

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FIG. 23D

IC50

clade virus 10E8- 1087 10E8- 1088 10E8- 1089 10E8- 1090 10E8- 1091 10E8- 1092 10E8- 1093 10E8-1094 10E8- 1095 A KER2008.12.SG3 11.1000 12.1000 12.0000 4.7500 >25 >25 >25 >25 >25 A RWO2O.2.SG3 0.0710 0.0680 0.0390 0.0550 0.0560 0.0520 0.0480 0.0550 0.0680 ACD 6095.V1.C10 <0.0003 <0.0003 <0.0003 0.0005 0.0004 <0.0003 <0.0003 <0.0003 <0.0003 AE CM244.ecl.SG3 0.0600 0.0300 0.0400 0.0380 0.0450 0.1240 0.0320 0.0480 0.0410 AE TH966.8 0.0060 0.0050 0.0040 0.0070 0.0050 0.0110 0.0030 0.0040 0.0040 B 6101.1 <0.0003 0.0005 0.0010 0.0020 0.0040 0.0030 0.0020 0.0020 0.0030 B CAAN.A2.SG3 0.1300 0.1320 0.0890 0.1100 0.1760 0.0700 0.1450 0.1010 0.1600 B PVO.04.SG3 0.0660 0.0620 0.0860 0.1250 0.0810 0.0880 0.0620 0.0350 0.0460 B YU2.DG.SG3 0.0490 0.0430 0.0330 0.0500 0.0360 0.0340 0.0240 0.0320 0.0470 C CNE31.SG3 0.0450 0.0390 0.0300 0.0540 0.0430 0.0240 0.0360 0.0330 0.0460 C ZM106.9.SG3 >25 >25 >25 >25 >25 >25 >25 >25 >25 C ZM215.8 0.0050 0.0040 0.0030 0.0050 0.0050 0.0020 0.0030 0.0040 0.0040 C ZM55.28a.SG3 0.0960 0.0880 0.0620 0.0960 0.0760 0.0420 0.0730 0.0590 0.0470 Median IC50 0.063 0.043 0.039 0.052 0.043 0.038 0.034 0.034 0.046 Geometric Mean 0.069 0.039 0.035 0.032 0.020 0.025 0.021 0.021 0.025

IC50

10E8-

clade virus 1022 A KER2008.12.SG3 >25 A RW020.2.SG3 0.1220 ACD 6095.V1.C10 0.0004 AE CM244.ecl.SG3 0.0530 AE TH966.8 0.0100 B 6101.1 0.0030 B CAAN.A2.SG3 0.2250 B PVO.04.SG3 0.0990 B YU2.DG.SG3 0.0940 C CNE31.SG3 0.0880 C ZM106.9.SG3 >25 C ZM215.8 0.0060 C ZM55.28a.SG3 0.1800 Median IC50 0.088 Geometric Mean 0.030

10E8- 1022 10E8- 1022 10E8v4 10E8v4 SlOOcF >25 >25 >50 >50 0.1290 0.0860 0.8650 0.0290 0.0004 <0.0003 0.0006 0.0000 0.0670 0.0360 0.7250 0.0460 0.0110 0.0060 0.0350 0.0010 0.0030 0.0020 0.0010 0.0000 0.2330 0.1560 2.2700 0.1450 0.1100 0.0510 2.5800 0.1000 0.0880 0.0570 2.0900 0.0810 0.0800 0.0850 1.1400 0.0500 >25 >25 >50 >50 0.0070 0.0050 0.0260 0.0080 0.1850 0.1430 2.0600 0.1400 0.080 0.054 0.865 0.046 0.031 0.032 0.187 0.009

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FIG. 23E

IC80

clade virus 10E8- 1065 10E8- 1068 10E8- 1069 10E8- 1070 10E8- 1072 10E8- 1073 10E8- 1074 10E8-1075 10E8- 1076 A KER2008.12.SG3 >25 >25 >20 >25 >25 9.9700 >25 >25 >25 A RWO2O.2.SG3 0.2340 0.4330 0.3310 0.5020 0.3570 0.2200 0.2820 0.2810 0.3450 ACD 6095.V1.C10 0.0020 0.0020 0.0010 0.0020 0.0010 0.0010 0.0010 0.0010 0.0020 AE CM244.ecl.SG3 0.1470 0.1770 0.1480 0.2700 0.2610 0.1790 0.2150 0.1790 0.3620 AE TH966.8 0.0290 0.0260 0.0190 0.0410 0.0250 0.0200 0.0230 0.0270 0.0410 B 6101.1 0.0070 0.0150 0.0080 0.0230 0.0070 0.0060 0.0070 0.0080 0.0090 B CAAN.A2.SG3 0.4770 0.6400 0.5510 0.7560 0.6390 0.3960 0.6770 0.5700 0.6570 B PVO.04.SG3 0.6240 0.7220 0.4330 1.3100 0.4480 0.8300 0.9600 0.8210 1.0500 B YU2.DG.SG3 0.3770 0.5750 0.3180 0.7780 0.2950 0.3020 0.3520 0.3890 0.5060 C CNE31.SG3 0.1540 0.2280 0.1380 0.2630 0.1640 0.1260 0.1430 0.1440 0.1850 C ZM106.9.SG3 >25 >25 >20 >25 >25 >25 >25 >25 >25 C ZM215.8 0.0190 0.0190 0.0130 0.0270 0.0170 0.0170 0.0150 0.0170 0.0220 C ZM55.28a.SG3 0.3360 0.3970 0.2690 0.4800 0.4320 0.3040 0.3710 0.3410 0.4810 Median IC80 0.154 0.228 0.148 0.270 0.261 0.200 0.215 0.179 0.345 Geometric Mean 0.084 0.110 0.072 0.147 0.085 0.110 0.085 0.085 0.118

clade IC80 virus 10E8- 1077 10E8- 1078 10E8- 1079 10E8- 1080 10E8- 1081 10E8- 1082 10E8- 1083 10E8-1084 10E8- 1086 A KER2008.12.SG3 >25 >25 >25 >25 >25 >25 >25 >25 >25 A RW020.2.SG3 0.3700 0.3850 0.3280 0.3640 0.1540 0.3450 1.4400 0.2750 0.2280 ACD 6095.V1.C10 0.0020 0.0020 0.0010 0.0020 0.0009 0.0020 0.0120 0.0010 0.0010 AE CM244.ecl.SG3 0.3040 0.3000 0.2580 0.1810 0.1240 0.1660 1.0100 0.3170 0.1850 AE TH966.8 0.0430 0.0350 0.0300 0.0240 0.0110 0.0250 0.1370 0.0180 0.0200 B 6101.1 0.0090 0.0080 0.0060 0.0220 0.0090 0.0280 0.6640 0.0130 0.0070 B CAAN.A2.SG3 0.5550 0.8030 0.8010 0.8760 0.5070 0.8840 3.2400 1.2400 0.5870 B PVO.04.SG3 0.9600 0.9840 0.9260 0.6340 0.2840 0.4990 4.9600 0.5010 0.4670 B YU2.DG.SG3 0.3910 0.4050 0.3900 0.5350 0.1230 0.5450 1.3400 0.4200 0.2750 C CNE31.SG3 0.1770 0.1750 0.1580 0.1990 0.0720 0.1740 0.7930 0.1630 0.1200 C ZM106.9.SG3 >25 >25 >25 >25 >25 >25 >25 >25 >25 C ZM215.8 0.0240 0.0140 0.0130 0.0160 0.0080 0.0150 0.1190 0.0210 0.0170 C ZM55.28a.SG3 0.4660 0.4460 0.4120 0.3550 0.1250 0.4160 1.3200 0.3760 0.3310 Median IC80 0.304 0.300 0.258 0.199 0.123 0.174 1.010 0.275 0.185 Geometric Mean 0.112 0.108 0.091 0.108 0.046 0.107 0.609 0.097 0.073

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FIG. 23F

IC80

clade virus 10E8- 1087 10E8- 1088 10E8- 1089 10E8- 1090 10E8- 1091 10E8- 1092 10E8- 1093 10E8-1094 10E8- 1095 A KER2008.12.SG3 >25 >25 >25 >25 >25 >25 >25 >25 >25 A RWO2O.2.SG3 0.2460 0.2760 0.1430 0.2220 0.2080 0.1890 0.1480 0.1840 0.2210 ACD 6095.V1.C10 0.0010 0.0010 0.0010 0.0020 0.0010 0.0010 0.0008 0.0007 0.0010 AE CM244.ecl.SG3 0.2520 0.1660 0.1730 0.1670 0.1670 0.5440 0.1190 0.1580 0.1320 AE TH966.8 0.0250 0.0220 0.0210 0.0310 0.0230 0.0450 0.0150 0.0140 0.0170 B 6101.1 0.0070 0.0070 0.0060 0.0080 0.0160 0.0130 0.0090 0.0130 0.0120 B CAAN.A2.SG3 0.6300 0.5980 0.3410 0.4690 0.6640 0.3900 0.7160 0.5530 0.6690 B PVO.04.SG3 0.4360 0.4940 0.4050 0.5710 0.4040 0.4230 0.2680 0.1730 0.2390 B YU2.DG.SG3 0.2820 0.2960 0.1700 0.2560 0.2130 0.1810 0.1370 0.1750 0.2100 C CNE31.SG3 0.1520 0.1350 0.0920 0.1300 0.1210 0.0720 0.1040 0.1060 0.1310 C ZM106.9.SG3 >25 >25 >25 >25 >25 >25 >25 >25 >25 C ZM215.8 0.0200 0.0150 0.0120 0.0190 0.0200 0.0100 0.0120 0.0090 0.0120 C ZM55.28a.SG3 0.3250 0.3640 0.1940 0.2870 0.2530 0.1280 0.1950 0.1790 0.1840 Median IC80 0.246 0.166 0.143 0.167 0.167 0.128 0.119 0.158 0.132 Geometric Mean 0.080 0.076 0.056 0.080 0.075 0.069 0.054 0.054 0.063

IC80

clade virus 10E8- 1022 10E8- 1022 10E8- 1022 10E8v4 10E8v4 SlOOcF A KER2008.12.SG3 >25 >25 >25 >50 >50 A RW020.2.SG3 0.4450 0.4370 0.3370 3.3100 0.2870 ACD 6095.V1.C10 0.0020 0.0020 0.0010 0.0010 0.0000 AE CM244.ecl.SG3 0.2340 0.2640 0.1710 2.7700 0.3180 AE TH966.8 0.0520 0.0480 0.0340 0.1920 0.0580 B 6101.1 0.0180 0.0160 0.0100 0.0560 0.0090 B CAAN.A2.SG3 1.0200 1.0800 0.8090 9.4600 0.9760 B PVO.04.SG3 0.5630 0.6290 0.3580 9.8600 0.6960 B YU2.DG.SG3 0.4300 0.4700 0.2980 9.2200 1.0100 C CNE31.SG3 0.2700 0.2290 0.1800 3.4700 0.2390 C ZM106.9.SG3 >25 >25 >25 >50 >50 C ZM215.8 0.0240 0.0270 0.0170 0.2670 0.0510 C ZM55.28a.SG3 0.5110 0.5870 0.4410 7.8600 0.5160 Median IC80 0.270 0.264 0.180 3.310 0.287 Geometric Mean 0.129 0.132 0.087 0.957 0.092

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FIG. 24

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Sequence_Listing SEQUENCE LISTING <110> The Government of the United States of America as

Represented by the Secretary, Department of Health and Human Services

The Trustees of Columbia University in the City of New York <120> NEUTRALIZING ANTIBODIES TO HIV-1 GP41 AND THEIR USE <130> 4239-94645-02 <150> US 62/250360 <151> 2015-11-03 <160> 86 <170> PatentIn version 3.5 <210> 1 <211> 131 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence

<400> 1 Gly Glu 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly Leu Val 10 Lys Pro Gly 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Asp Asn Ala 20 25 30 Trp Met Thr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Thr Gly Pro Gly Glu Gly Trp Ser Val Asp Tyr Ala Ala 50 55 60 Pro Val Glu Gly Arg Phe Thr Ile Ser Arg Leu Asn Ser Ile Asn Phe 65 70 75 80 Leu Tyr Leu Glu Met Asn Asn Leu Arg Met Glu Asp Ser Gly Leu Tyr 85 90 95 Phe Cys Ala Arg Thr Gly Lys Tyr Tyr Asp Phe Trp Ser Gly Tyr Pro 100 105 110 Pro Gly Glu Glu Tyr Phe Gln Asp Trp Gly Arg Gly Thr Leu Val Thr 115 120 125

Val Ser Ser 130 <210> 2 <211> 109 <212> PRT <213> Artificial Sequence

Page 1

Sequence_Listing <220>

<223> Variant Antibody Sequence <400> 2

Ser Tyr Glu 1 Leu Thr Gln Glu Thr Gly Val Ser Val Ala Leu Gly 15 Arg 5 10 Thr Val Thr Ile Thr Cys Arg Gly Asp Ser Leu Arg Ser His Tyr Ala 20 25 30 Ser Trp Tyr Gln Lys Lys Pro Gly Gln Ala Pro Ile Leu Leu Phe Tyr 35 40 45 Gly Lys Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ala Ser Gly Asn Arg Ala Ser Leu Thr Ile Ser Gly Ala Gln Ala Glu 65 70 75 80 Asp Asp Ala Glu Tyr Tyr Cys Ser Ser Arg Asp Lys Ser Gly Ser Arg 85 90 95 Leu Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 3 <211> 131 <212> PRT <213> Artificial Sequence <220> <223> Variant Antibody Sequence <400> 3 Glu Val Arg Leu Ala Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Asp Asn Ala 20 25 30 Trp Met Thr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Thr Gly Pro Gly Glu Gly Trp Ser Val Asp Tyr Ala Ala 50 55 60 Pro Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Tyr Lys Asn Thr 65 70 75 80 Leu Tyr Leu Glu Met Asn Asn Leu Arg Thr Glu Asp Ser Gly Leu Tyr 85 90 95

Page 2

Sequence_Listing

Phe Cys Ala Arg 100 Thr Gly Lys Tyr Tyr Asp Phe Trp Ser Gly Tyr Pro 105 110 Pro Gly Glu Glu Tyr Phe Gln Asp Trp Gly Arg Gly Thr Leu Val Ile 115 120 125 Val Ser Ser

130 <210> 4 <211> 109 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 4

Ser Tyr Glu 1 Leu Thr Gln Asp Thr Gly Val Ser Val Ala Leu Gly 15 Arg 5 10 Thr Val Thr Ile Thr Cys Arg Gly Asp Ser Leu Arg Ser His Tyr Ala 20 25 30 Ser Trp Tyr Gln Lys Lys Pro Gly Gln Ala Pro Val Leu Leu Phe Tyr 35 40 45 Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ala Ser Gly Asn Arg Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Arg Asp Lys Ser Gly Ser Arg 85 90 95

Leu Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 5 <211> 131 <212> PRT

<213> Artificial Sequence <220>

<223> Variant Antibody Sequence

<400> 5 Glu Val Arg Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Asp Asn Ala 20 25 30

Page 3

Trp Met Thr Trp Val Arg Gln Pro 40 Sequence_Li sting Pro Gly Lys Gly Leu 45 Glu Trp Val 35 Gly Arg Ile Thr Gly Pro Gly Glu Gly Trp Ser Val Asp Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr 65 70 75 80 Leu Tyr Leu Glu Met Asn Asn Val Arg Thr Glu Asp Thr Gly Tyr Tyr 85 90 95 Phe Cys Ala Arg Thr Gly Lys Tyr Tyr Asp Phe Trp Ser Gly Tyr Pro 100 105 110 Pro Gly Glu Glu Tyr Phe Gln Asp Trp Gly Gln Gly Thr Leu Val Ile 115 120 125 Val Ser Ser

130 <210> 6 <211> 109 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 6 Ala Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Lys Gln 1 5 10 15 Thr Val Thr Ile Thr Cys Arg Gly Asp Ser Leu Arg Ser His Tyr Ala 20 25 30 Ser Trp Tyr Gln Lys Lys Pro Gly Gln Ala Pro Val Leu Leu Phe Tyr 35 40 45 Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ala Ser Gly Asn Arg Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Arg Asp Lys Ser Gly Ser Arg 85 90 95 Leu Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 <210> 7 <211> 8

Page 4

Sequence_Listing <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 7

Gly Phe Asp Phe Asp Asn Ala Trp 1 5 <210> 8 <211> 10 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 8

Ile Thr Gly Pro Gly Glu Gly Trp Ser Val 1 5 10 <210> 9 <211> 22 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 9

Ala Arg Thr Gly Lys Tyr Tyr Asp Phe Trp Ser Gly Tyr Pro Pro Gly 1 5 10 15

Glu Glu Tyr Phe Gln Asp 20 <210> 10 <211> 6 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 10

Ser Leu Arg Ser His Tyr 1 5 <210> 11 <211> 3 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 11

Page 5

Sequence_Listing

Gly Lys Asn 1 <210> 12 <211> 12 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 12

Ser Ser Arg Asp Lys Ser Gly Ser Arg Leu Ser Val 1 5 10 <210> 13 <211> 131 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 13

Glu Val Arg Leu Ala Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Asp Asn Ala 20 25 30 Trp Met Thr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Thr Gly Pro Gly Glu Gly Trp Ser Val Asp Tyr Ala Ala 50 55 60 Pro Val Glu Gly Arg Phe Thr Ile Ser Arg Leu Asn Tyr Ile Asn Phe 65 70 75 80 Leu Tyr Leu Glu Met Asn Asn Leu Arg Met Glu Asp Ser Gly Leu Tyr 85 90 95 Phe Cys Ala Arg Thr Gly Lys Tyr Tyr Asp Phe Trp Ser Gly Tyr Pro 100 105 110 Pro Gly Glu Glu Tyr Phe Gln Asp Trp Gly Arg Gly Thr Leu Val Ile 115 120 125 Val Ser Ser 130

<210> 14 <211> 131 <212> PRT <213> Artificial Sequence

Page 6

Sequence_Listing <220>

<223> Variant Antibody Sequence <400> 14

Glu 1 Val Arg Leu Val 5 Glu Ser Gly Gly Gly Leu Val 10 Lys Pro Gly Gly 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asn Phe Asp Asp Ala 20 25 30 Trp Met Thr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Ser Gly Pro Gly Glu Gly Trp Ser Val Asp Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Leu Asn Ser Ile Asn Phe 65 70 75 80 Leu Tyr Leu Glu Met Asn Asn Val Arg Thr Glu Asp Thr Gly Tyr Tyr 85 90 95 Phe Cys Ala Arg Thr Gly Lys His Tyr Asp Phe Trp Ser Gly Tyr Pro 100 105 110 Pro Gly Glu Glu Tyr Phe Gln Asp Trp Gly Gln Gly Thr Leu Val Ile 115 120 125 Val Ser Ser 130 <210> 15 <211> 131 <212> PRT <213> , Artificial Sequence <220> <223> ' Variant Antibody Sequence <400> 15 Glu Val Arg Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asn Phe Asp Asp Ala 20 25 30 Trp Met Thr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Ser Gly Pro Gly Glu Gly Trp Ser Val Asp Tyr Ala Glu

50 55 60

Page 7

Sequence_Listing

Ser 65 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr 80 70 75 Leu Tyr Leu Glu Met Asn Asn Val Arg Thr Glu Asp Thr Gly Tyr Tyr 85 90 95 Phe Cys Ala Arg Thr Gly Lys His Tyr Asp Phe Trp Ser Gly Tyr Pro 100 105 110 Pro Gly Glu Glu Tyr Phe Gln Asp Trp Gly Gln Gly Thr Leu Val Ile 115 120 125 Val Ser Ser 130 <210> 16 <211> 131 <212> PRT <213> Artificial Sequence <220> <223> Variant Antibody Sequence <400> 16 Glu Val Arg Leu Val Glu Ser Gly Gly Arg Leu Val Arg Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asn Phe Asp Asn Ala 20 25 30 Trp Met Thr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Thr Gly Pro Gly Glu Gly Trp Ser Val Asp Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Met Asn Ser Ile Asn Phe 65 70 75 80 Phe Tyr Leu Glu Met Asn Asn Leu Lys Ile Glu Asp Thr Gly Leu Tyr 85 90 95 Phe Cys Ala Arg Thr Gly Lys His Tyr Ala Phe Trp Gly Gly Tyr Pro 100 105 110 Pro Gly Glu Glu Tyr Leu Glu Asp Trp Gly Gln Gly Thr Leu Val Ile 115 120 125

Val Ser Ser 130 <210> 17 <211> 131

Page 8

Sequence_Listing <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 17

Glu 1 Val Arg Leu Val 5 Glu Ser Gly Gly Arg Leu Val 10 Arg Pro Gly Gly 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asn Phe Asp Asn Ala 20 25 30 Trp Met Thr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Thr Gly Pro Gly Glu Gly Trp Ser Val Asp Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Tyr Lys Asn Thr 65 70 75 80 Phe Tyr Leu Glu Met Asn Asn Leu Lys Thr Glu Asp Thr Gly Leu Tyr 85 90 95 Phe Cys Ala Arg Thr Gly Lys His Tyr Ala Phe Trp Gly Gly Tyr Pro 100 105 110 Pro Gly Glu Glu Tyr Leu Glu Asp Trp Gly Gln Gly Thr Leu Val Ile 115 120 125 Val Ser Ser 130 <210> 18 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Variant Antibody Sequence <400> 18 Ala Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Lys Gln 1 5 10 15 Thr Val Thr Ile Thr Cys Arg Gly Asp Ser Leu Arg Ser His Tyr Val 20 25 30 Ser Trp Tyr Gln Lys Lys Pro Gly Gln Ala Pro Val Leu Val Phe Tyr 35 40 45 Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Page 9

Sequence_Listing

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Ala Gly Ala Gln Ala Glu 65 70 75 80 Asp Asp Ala Asp Tyr Tyr Cys Ser Ser Arg Asp Lys Ser Gly Ser Arg 85 90 95 Leu Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 <210> 19 <211> 109 <212> PRT <213> Artificial Sequence

<220> <223> Variant Antibody Sequence <400> 19 Ala Ser Glu Leu Thr Gln Asp Pro Thr Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Val Thr Ile Thr Cys Arg Gly Asp Ser Leu Arg Asn Tyr Tyr Thr 20 25 30 Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Leu Ile Tyr 35 40 45 Pro Lys His Asn Arg Pro Pro Gly Ile Ser Asp Arg Phe Ser Ala Ser 50 55 60 Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Thr Glu 65 70 75 80 Asp Glu Gly Asp Tyr Tyr Cys Ser Ser Arg Asp Lys Ser Gly Ser Arg 85 90 95 Leu Val Thr Phe Gly Arg Gly Thr Lys Leu Thr Val Val 100 105

<210> 20 <211> 1443 <212> DNA <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 20

atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattctgaa 60 gtgcggctgg ctgagagcgg cggggggctg gtcaaacctg gcgggtcact gcggctgtcc 120 tgttctgcct ccggcttcga ttttgataac gcatggatga catgggtgcg acagccacct 180 ggaaaggggc tggagtgggt cggcagaatc actggacctg gcgaagggtg gtctgtggac Page 10 240

Sequence_Listing

tacgcagctc cagtcgaggg acgattcacc attagtagag ataactacaa gaatacactg 300 tatctggaga tgaacaatct gaggactgaa gacagcggcc tgtatttctg cgcccgcacc 360 gggaaatact atgatttttg gtctgggtac ccacccggag aggaatattt tcaggactgg 420 ggacggggca ccctggtgat cgtcagctcc gcgtcgacca agggcccatc ggtcttcccc 480 ctggcaccct cctccaagag cacctctggg ggcacagcgg ccctgggctg cctggtcaag 540 gactacttcc ccgaaccggt gacggtgtcg tggaactcag gcgccctgac cagcggcgtg 600 cacaccttcc cggctgtcct acagtcctca ggactctact ccctcagcag cgtggtgacc 660 gtgccctcca gcagcttggg cacccagacc tacatctgca acgtgaatca caagcccagc 720 aacaccaagg tggacaagaa agttgagccc aaatcttgtg acaaaactca cacatgccca 780 ccgtgcccag cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc 840 aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc 900 cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc 960 aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc 1020 gtcctgcacc aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc 1080 ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag 1140 gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgacctgc 1200 ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg 1260 gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac 1320 agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg 1380 atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaaa 1440

tga 1443 <210> 21 <211> 1440

<212> DNA <213> Artificial Sequence <220> <223> Variant Antibody Sequence <400> 21 atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattctgag 60 gttagactgg tggagtcagg aggggggctt gtgaagcccg gtgggtctct ccgcctgagc 120 tgttctgcct ccggctttga tttcgataac gcctggatga cctgggtcag gcagcctcca 180 ggtaagggac tggagtgggt gggaagaatc acaggtccag gcgagggctg gtccgtggac 240 tacgcggaat ctgttaaagg gcggtttaca atctcaaggg acaataccaa gaataccttg 300 tatttggaga tgaacaacgt gagaactgaa gacaccggat attacttctg tgccagaaca 360 ggcaaatact acgacttctg gtccggctat ccccctggcg aggaatattt tcaagactgg 420 ggtcagggaa cccttgttat cgtgtcctcc gcgtcgacca agggcccatc ggtcttcccc 480

Page 11

Sequence_Listing

ctggcaccct cctccaagag cacctctggg ggcacagcgg ccctgggctg cctggtcaag 540 gactacttcc ccgaaccggt gacggtgtcg tggaactcag gcgccctgac cagcggcgtg 600 cacaccttcc cggctgtcct acagtcctca ggactctact ccctcagcag cgtggtgacc 660 gtgccctcca gcagcttggg cacccagacc tacatctgca acgtgaatca caagcccagc 720 aacaccaagg tggacaagaa agttgagccc aaatcttgtg acaaaactca cacatgccca 780 ccgtgcccag cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc 840 aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc 900 cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc 960 aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc 1020 gtcctgcacc aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc 1080 ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag 1140 gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgacctgc 1200 ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg 1260 gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac 1320 agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg 1380 atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaaa 1440

<210> 22 <211> 461 <212> PRT <213> Artificial Sequence

<220> <223> Variant Antibody Sequence <400> 22 Glu Val Arg Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Asp Asn Ala 20 25 30 Trp Met Thr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Thr Gly Pro Gly Glu Gly Trp Ser Val Asp Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr 65 70 75 80 Leu Tyr Leu Glu Met Asn Asn Val Arg Thr Glu Asp Thr Gly Tyr Tyr

85 90 95

Page 12

Phe Cys Ala Sequence_Listing Arg 100 Thr Gly Lys Tyr Tyr 105 Asp Phe Trp Ser Gly 110 Tyr Pro Pro Gly Glu 115 Glu Tyr Phe Gln Asp 120 Trp Gly Gln Gly Thr 125 Leu Val Ile Val Ser Ser 130 Ala Ser Thr Lys 135 Gly Pro Ser Val Phe 140 Pro Leu Ala Pro Ser 145 Ser Lys Ser Thr Ser 150 Gly Gly Thr Ala Ala 155 Leu Gly Cys Leu Val 160 Lys Asp Tyr Phe Pro 165 Glu Pro Val Thr Val 170 Ser Trp Asn Ser Gly 175 Ala Leu Thr Ser Gly 180 Val His Thr Phe Pro 185 Ala Val Leu Gln Ser 190 Ser Gly Leu Tyr Ser 195 Leu Ser Ser Val Val 200 Thr Val Pro Ser Ser 205 Ser Leu Gly Thr Gln Thr 210 Tyr Ile Cys Asn 215 Val Asn His Lys Pro 220 Ser Asn Thr Lys Val 225 Asp Lys Lys Val Glu 230 Pro Lys Ser Cys Asp 235 Lys Thr His Thr Cys 240 Pro Pro Cys Pro Ala 245 Pro Glu Leu Leu Gly 250 Gly Pro Ser Val Phe 255 Leu Phe Pro Pro Lys 260 Pro Lys Asp Thr Leu 265 Met Ile Ser Arg Thr 270 Pro Glu Val Thr Cys 275 Val Val Val Asp Val 280 Ser His Glu Asp Pro 285 Glu Val Lys Phe Asn Trp 290 Tyr Val Asp Gly 295 Val Glu Val His Asn 300 Ala Lys Thr Lys Pro 305 Arg Glu Glu Gln Tyr 310 Asn Ser Thr Tyr Arg 315 Val Val Ser Val Leu 320 Thr Val Leu His Gln 325 Asp Trp Leu Asn Gly 330 Lys Glu Tyr Lys Cys 335 Lys Val Ser Asn Lys 340 Ala Leu Pro Ala Pro 345 Ile Glu Lys Thr Ile 350 Ser Lys Ala Lys Gly 355 Gln Pro Arg Glu Pro 360 Gln Val Tyr Thr Leu 365 Pro Pro Ser

Page 13

Sequence_Listing

Arg Asp Glu 370 Leu Thr Lys Asn 375 Gln Val Ser Leu Thr 380 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 385 390 395 400 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 405 410 415 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 420 425 430 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 435 440 445 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455 460 <210> 23 <211> 461 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 23 Glu Val Arg Leu Ala Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Asp Asn Ala 20 25 30 Trp Met Thr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Thr Gly Pro Gly Glu Gly Trp Ser Val Asp Tyr Ala Ala 50 55 60 Pro Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Tyr Lys Asn Thr 65 70 75 80 Leu Tyr Leu Glu Met Asn Asn Leu Arg Thr Glu Asp Ser Gly Leu Tyr 85 90 95 Phe Cys Ala Arg Thr Gly Lys Tyr Tyr Asp Phe Trp Ser Gly Tyr Pro 100 105 110 Pro Gly Glu Glu Tyr Phe Gln Asp Trp Gly Arg Gly Thr Leu Val Ile 115 120 125 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 130 135 140

Page 14

Sequence_Listing

Ser 145 Ser Lys Ser Thr Ser Gly Gly Thr 150 Ala Ala Leu Gly Cys 155 Leu Val 160 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 165 170 175 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 180 185 190 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 195 200 205 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 210 215 220 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 225 230 235 240 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 245 250 255 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 260 265 270 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 275 280 285 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 290 295 300 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 305 310 315 320 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 325 330 335 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 340 345 350 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 355 360 365 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 370 375 380 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 385 390 395 400 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 405 410 415

Page 15

Sequence_Listing

Ser Phe Phe Leu Tyr Ser 420 Lys Leu Thr 425 Val Asp Lys Ser Arg 430 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 435 440 445 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455 460 <210> 24 <211> 648 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 24

Thr Cys Ala Thr Ala Cys Gly Ala Ala Cys Thr Gly Ala Cys Thr Cys 1 5 10 15 Ala Gly Gly Ala Cys Ala Cys Thr Gly Gly Cys Gly Thr Cys Thr Cys 20 25 30 Thr Gly Thr Gly Gly Cys Ala Cys Thr Gly Gly Gly Gly Ala Gly Gly 35 40 45 Ala Cys Thr Gly Thr Gly Ala Cys Thr Ala Thr Thr Ala Cys Thr Thr 50 55 60 Gly Cys Cys Gly Ala Gly Gly Cys Gly Ala Cys Thr Cys Ala Cys Thr 65 70 75 80 Gly Cys Gly Gly Ala Gly Cys Cys Ala Cys Thr Ala Cys Gly Cys Thr 85 90 95 Thr Cys Cys Thr Gly Gly Thr Ala Thr Cys Ala Gly Ala Ala Gly Ala 100 105 110 Ala Ala Cys Cys Cys Gly Gly Cys Cys Ala Gly Gly Cys Ala Cys Cys 115 120 125 Thr Gly Thr Gly Cys Thr Gly Cys Thr Gly Thr Thr Cys Thr Ala Cys 130 135 140 Gly Gly Ala Ala Ala Gly Ala Ala Cys Ala Ala Thr Ala Gly Gly Cys 145 150 155 160 Cys Ala Thr Cys Thr Gly Gly Cys Ala Thr Cys Cys Cys Cys Gly Ala 165 170 175

Page 16

Cys Cys Gly Cys Thr Thr Sequence_Listing Thr Thr Cys 185 Thr Gly Gly Cys Ala 190 Gly Thr 180 Gly Cys Ala Thr Cys Ala Gly Gly Gly Ala Ala Cys Cys Gly Ala Gly 195 200 205 Cys Cys Ala Gly Thr Cys Thr Gly Ala Cys Cys Ala Thr Thr Ala Cys 210 215 220 Cys Gly Gly Cys Gly Cys Cys Cys Ala Gly Gly Cys Thr Gly Ala Gly 225 230 235 240 Gly Ala Cys Gly Ala Ala Gly Cys Cys Gly Ala Thr Thr Ala Cys Thr 245 250 255 Ala Thr Thr Gly Cys Ala Gly Cys Thr Cys Cys Cys Gly Gly Gly Ala 260 265 270 Thr Ala Ala Gly Ala Gly Cys Gly Gly Cys Thr Cys Cys Ala Gly Ala 275 280 285 Cys Thr Gly Ala Gly Cys Gly Thr Gly Thr Thr Cys Gly Gly Ala Gly 290 295 300 Gly Ala Gly Gly Ala Ala Cys Thr Ala Ala Ala Cys Thr Gly Ala Cys 305 310 315 320 Cys Gly Thr Cys Cys Thr Cys Ala Gly Thr Cys Ala Gly Cys Cys Cys 325 330 335 Ala Ala Gly Gly Cys Thr Gly Cys Cys Cys Cys Cys Thr Cys Gly Gly 340 345 350 Thr Cys Ala Cys Thr Cys Thr Gly Thr Thr Cys Cys Cys Gly Cys Cys 355 360 365 Cys Thr Cys Gly Ala Gly Thr Gly Ala Gly Gly Ala Gly Cys Thr Thr 370 375 380 Cys Ala Ala Gly Cys Cys Ala Ala Cys Ala Ala Gly Gly Cys Cys Ala 385 390 395 400 Cys Ala Cys Thr Gly Gly Thr Gly Thr Gly Thr Cys Thr Cys Ala Thr 405 410 415 Ala Ala Gly Thr Gly Ala Cys Thr Thr Cys Thr Ala Cys Cys Cys Gly 420 425 430 Gly Gly Ala Gly Cys Cys Gly Thr Gly Ala Cys Ala Gly Thr Gly Gly 435 440 445

Page 17

Cys Cys Thr Gly Gly Sequence_Listing Ala Ala Gly Gly Cys Ala Gly Ala Thr Ala Gly 450 455 460 Cys Ala Gly Cys Cys Cys Cys Gly Thr Cys Ala Ala Gly Gly Cys Gly 465 470 475 480 Gly Gly Ala Gly Thr Gly Gly Ala Gly Ala Cys Cys Ala Cys Cys Ala 485 490 495 Cys Ala Cys Cys Cys Thr Cys Cys Ala Ala Ala Cys Ala Ala Ala Gly 500 505 510 Cys Ala Ala Cys Ala Ala Cys Ala Ala Gly Thr Ala Cys Gly Cys Gly 515 520 525 Gly Cys Cys Ala Gly Cys Ala Gly Cys Thr Ala Cys Cys Thr Gly Ala 530 535 540 Gly Cys Cys Thr Gly Ala Cys Gly Cys Cys Thr Gly Ala Gly Cys Ala 545 550 555 560 Gly Thr Gly Gly Ala Ala Gly Thr Cys Cys Cys Ala Cys Ala Gly Ala 565 570 575 Ala Gly Cys Thr Ala Cys Ala Gly Cys Thr Gly Cys Cys Ala Gly Gly 580 585 590 Thr Cys Ala Cys Gly Cys Ala Thr Gly Ala Ala Gly Gly Gly Ala Gly 595 600 605 Cys Ala Cys Cys Gly Thr Gly Gly Ala Gly Ala Ala Gly Ala Cys Ala 610 615 620 Gly Thr Gly Gly Cys Cys Cys Cys Thr Ala Cys Ala Gly Ala Ala Thr 625 630 635 640 Gly Thr Thr Cys Ala Thr Ala Gly 645

<210> 25 <211> 678 <212> PRT <213> Artificial Sequence <220> <223> Variant Antibody Sequence

<400> 25

Ala Cys Cys Ala Cys Cys Ala Thr Gly Gly Gly Ala Thr Gly Gly Thr 1 5 10 15 Cys Ala Thr Gly Thr Ala Thr Cys Ala Thr Cys Cys Thr Thr Thr Thr

20 25 30

Page 18

Sequence_Listing

Thr Cys

Ala Cys 50

Cys Ala 65

Gly Gly

Thr Ala 35

Cys Gly

Thr Cys

Ala Cys

Gly Thr

Gly Gly 100

Cys Thr

Gly Thr 115

Cys Cys 130

Gly Ala

Cys Gly 145

Gly Ala

Cys Cys

Thr Gly

Ala Cys

Cys Cys 180

Gly Thr

Gly Cys 195

Gly Ala 210

Ala Ala

Ala Thr 225

Cys Thr

Cys Gly

Cys Thr

Cys Ala

Thr Cys 260

Cys Ala

Gly Thr 275

Gly Gly 290

Cys Gly

Gly

Cys

Gly

165

Gly

Thr

Gly

Thr

245

Ala

Cys

Thr Ala

Thr Thr 55

Gly Ala 70

Cys Thr

Ala Cys

Gly Cys 135

Cys Cys 150

Thr Ala

Gly Cys

Ala Ala 215

Gly Cys 230

Thr Thr

Gly Gly

Thr Gly

Cys Cys 295

Gly Cys 40

Cys Thr

Ala Cys

Gly Cys

Thr Gly 105

Thr Ala 120

Gly Ala

Ala Cys

Thr Cys

Cys Ala 185

Thr Gly 200

Cys Ala

Ala Thr

Cys Thr

Gly Ala 265

Ala Cys 280

Ala Gly

Ala Ala

Gly Thr

Thr Gly 75

Cys Gly 90

Ala Ala

Thr Thr

Cys Thr

Thr Ala 155

Ala Gly 170

Gly Gly

Thr Thr

Ala Thr

Cys Cys 235

Gly Gly 250

Ala Cys

Cys Ala

Gly Cys

Cys Thr 45

Gly Ala 60

Ala Cys

Thr Cys

Gly Cys

Ala Cys 125

Cys Ala 140

Cys Gly

Ala Ala

Cys Ala

Cys Thr 205

Ala Gly 220

Cys Cys

Cys Ala

Cys Gly

Thr Thr 285

Thr Gly 300

Gly

Cys

Thr

Ala

110

Thr

Cys

Gly

Cys

190

Ala

Gly

Ala

270

Ala

Cys Ala

Cys Gly

Cys Ala 80

Cys Thr 95

Gly Ala

Thr Gly

Thr Thr 160

Ala Ala 175

Cys Thr

Cys Gly

Cys Cys

Ala Cys 240

Thr Gly 255

Gly Cys

Cys Cys

Gly Gly

Page 19

Sequence_Listing

Ala Cys Gly Ala Ala Gly Cys Cys Gly Ala Thr Thr Ala Cys Thr Ala 305 310 315 320 Thr Thr Gly Cys Ala Gly Cys Thr Cys Cys Cys Gly Gly Gly Ala Thr 325 330 335 Ala Ala Gly Ala Gly Cys Gly Gly Cys Thr Cys Cys Ala Gly Ala Cys 340 345 350 Thr Gly Ala Gly Cys Gly Thr Gly Thr Thr Cys Gly Gly Ala Gly Gly 355 360 365 Ala Gly Gly Ala Ala Cys Thr Ala Ala Ala Cys Thr Gly Ala Cys Cys 370 375 380 Gly Thr Cys Cys Thr Cys Ala Gly Thr Cys Ala Gly Cys Cys Cys Ala 385 390 395 400 Ala Gly Gly Cys Thr Gly Cys Cys Cys Cys Cys Thr Cys Gly Gly Thr 405 410 415 Cys Ala Cys Thr Cys Thr Gly Thr Thr Cys Cys Cys Gly Cys Cys Cys 420 425 430 Thr Cys Gly Ala Gly Thr Gly Ala Gly Gly Ala Gly Cys Thr Thr Cys 435 440 445 Ala Ala Gly Cys Cys Ala Ala Cys Ala Ala Gly Gly Cys Cys Ala Cys 450 455 460 Ala Cys Thr Gly Gly Thr Gly Thr Gly Thr Cys Thr Cys Ala Thr Ala 465 470 475 480 Ala Gly Thr Gly Ala Cys Thr Thr Cys Thr Ala Cys Cys Cys Gly Gly 485 490 495 Gly Ala Gly Cys Cys Gly Thr Gly Ala Cys Ala Gly Thr Gly Gly Cys 500 505 510 Cys Thr Gly Gly Ala Ala Gly Gly Cys Ala Gly Ala Thr Ala Gly Cys 515 520 525 Ala Gly Cys Cys Cys Cys Gly Thr Cys Ala Ala Gly Gly Cys Gly Gly 530 535 540 Gly Ala Gly Thr Gly Gly Ala Gly Ala Cys Cys Ala Cys Cys Ala Cys 545 550 555 560 Ala Cys Cys Cys Thr Cys Cys Ala Ala Ala Cys Ala Ala Ala Gly Cys

565 570 575

Page 20

Sequence_Listing

Ala Ala Cys Ala Ala Cys Ala Ala Gly Thr Ala Cys Gly Cys Gly Gly 580 585 590 Cys Cys Ala Gly Cys Ala Gly Cys Thr Ala Cys Cys Thr Gly Ala Gly 595 600 605 Cys Cys Thr Gly Ala Cys Gly Cys Cys Thr Gly Ala Asn Cys Ala Gly 610 615 620 Thr Gly Gly Ala Ala Asn Gly Thr Cys Cys Cys Ala Cys Ala Gly Ala 625 630 635 640 Ala Gly Cys Thr Ala Cys Ala Gly Cys Thr Gly Cys Cys Ala Gly Gly 645 650 655 Thr Cys Ala Cys Gly Cys Ala Thr Gly Ala Ala Gly Gly Gly Ala Gly 660 665 670 Cys Ala Cys Cys Gly Thr 675 <210> 26 <211> 856 <212> PRT <213> Human immunodeficiency virus type 1 <400> 26 Met Arg Val Lys Glu Lys Tyr Gln His Leu Trp Arg Trp Gly Trp Arg 1 5 10 15 Trp Gly Thr Met Leu Leu Gly Met Leu Met Ile Cys Ser Ala Thr Glu 20 25 30 Lys Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala 35 40 45 Thr Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu 50 55 60 Val His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn 65 70 75 80 Pro Gln Glu Val Val Leu Val Asn Val Thr Glu Asn Phe Asn Met Trp 85 90 95 Lys Asn Asp Met Val Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp 100 105 110 Asp Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Ser 115 120 125

Page 21

Sequence_Listing

Leu Lys Cys Thr Asp 130 Leu Lys Asn Asp Thr Asn Thr Asn Ser Ser Ser 135 140 Gly Arg Met Ile Met Glu Lys Gly Glu Ile Lys Asn Cys Ser Phe Asn 145 150 155 160 Ile Ser Thr Ser Ile Arg Gly Lys Val Gln Lys Glu Tyr Ala Phe Phe 165 170 175 Tyr Lys Leu Asp Ile Ile Pro Ile Asp Asn Asp Thr Thr Ser Tyr Lys 180 185 190 Leu Thr Ser Cys Asn Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Val 195 200 205 Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Ala 210 215 220 Ile Leu Lys Cys Asn Asn Lys Thr Phe Asn Gly Thr Gly Pro Cys Thr 225 230 235 240 Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro Val Val Ser 245 250 255 Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Val Val Ile 260 265 270 Arg Ser Val Asn Phe Thr Asp Asn Ala Lys Thr Ile Ile Val Gln Leu 275 280 285 Asn Thr Ser Val Glu Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg 290 295 300 Lys Arg Ile Arg Ile Gln Arg Gly Pro Gly Arg Ala Phe Val Thr Ile 305 310 315 320 Gly Lys Ile Gly Asn Met Arg Gln Ala His Cys Asn Ile Ser Arg Ala 325 330 335 Lys Trp Asn Asn Thr Leu Lys Gln Ile Ala Ser Lys Leu Arg Glu Gln 340 345 350 Phe Gly Asn Asn Lys Thr Ile Ile Phe Lys Gln Ser Ser Gly Gly Asp 355 360 365 Pro Glu Ile Val Thr His Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr 370 375 380 Cys Asn Ser Thr Gln Leu Phe Asn Ser Thr Trp Phe Asn Ser Thr Trp 385 390 395 400

Page 22

Sequence_Listing

Ser Thr Glu Gly Ser 405 Asn Asn Thr Glu Gly Ser Asp Thr 410 Ile Thr 415 Leu Pro Cys Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Lys Val Gly Lys 420 425 430 Ala Met Tyr Ala Pro Pro Ile Ser Gly Gln Ile Arg Cys Ser Ser Asn 435 440 445 Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly Asn Ser Asn Asn Glu 450 455 460 Ser Glu Ile Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp Arg 465 470 475 480 Ser Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile Glu Pro Leu Gly Val 485 490 495 Ala Pro Thr Lys Ala Lys Arg Arg Val Val Gln Arg Glu Lys Arg Ala 500 505 510 Val Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser 515 520 525 Thr Met Gly Ala Ala Ser Met Thr Leu Thr Val Gln Ala Arg Gln Leu 530 535 540 Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu 545 550 555 560 Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu 565 570 575 Gln Ala Arg Ile Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu 580 585 590 Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala Val 595 600 605 Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn 610 615 620 His Thr Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser 625 630 635 640 Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn 645 650 655 Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp 660 665 670

Page 23

Sequence_Listing

Phe Asn Ile Thr Asn Trp Leu Trp 680 Tyr Ile Lys Leu Phe 685 Ile Met Ile 675 Val Gly Gly Leu Val Gly Leu Arg Ile Val Phe Ala Val Leu Ser Ile 690 695 700 Val Asn Arg Val Arg Gln Gly Tyr Ser Pro Leu Ser Phe Gln Thr His 705 710 715 720 Leu Pro Thr Pro Arg Gly Pro Asp Arg Pro Glu Gly Ile Glu Glu Glu 725 730 735 Gly Gly Glu Arg Asp Arg Asp Arg Ser Ile Arg Leu Val Asn Gly Ser 740 745 750 Leu Ala Leu Ile Trp Asp Asp Leu Arg Ser Leu Cys Leu Phe Ser Tyr 755 760 765 His Arg Leu Arg Asp Leu Leu Leu Ile Val Thr Arg Ile Val Glu Leu 770 775 780 Leu Gly Arg Arg Gly Trp Glu Ala Leu Lys Tyr Trp Trp Asn Leu Leu 785 790 795 800 Gln Tyr Trp Ser Gln Glu Leu Lys Asn Ser Ala Val Ser Leu Leu Asn 805 810 815 Ala Thr Ala Ile Ala Val Ala Glu Gly Thr Asp Arg Val Ile Glu Val 820 825 830 Val Gln Gly Ala Cys Arg Ala Ile Arg His Ile Pro Arg Arg Ile Arg 835 840 845 Gln Gly Leu Glu Arg Ile Leu Leu

850 855 <210> 27 <211> 121 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 27 Gln Val Gln Leu Val Gln Ser Gly Gly Gln Met Lys Lys Pro Gly Glu 1 5 10 15 Ser Met Arg Ile Ser Cys Arg Ala Ser Gly Tyr Glu Phe Ile Asp Cys 20 25 30 Thr Leu Asn Trp Ile Arg Leu Ala Pro Gly Lys Arg Pro Glu Trp Met

Page 24

35 Sequence_Listing 40 45 Gly Trp Leu Lys Pro Arg Gly Gly Ala Val Asn Tyr Ala Arg Pro Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Val Tyr Ser Asp Thr Ala Phe 65 70 75 80 Leu Glu Leu Arg Ser Leu Thr Val Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95 Thr Arg Gly Lys Asn Cys Asp Tyr Asn Trp Asp Phe Glu His Trp Gly 100 105 110 Arg Gly Thr Pro Val Ile Val Ser Ser 115 120

<210> 28 <211> 103 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 28

Glu 1 Ile Val Leu Thr Gln Ser 5 Pro Gly Thr 10 Leu Ser Leu Ser Pro 15 Gly Glu Thr Ala Ile Ile Ser Cys Arg Thr Ser Gln Tyr Gly Ser Leu Ala 20 25 30 Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Val Ile Tyr Ser 35 40 45 Gly Ser Thr Arg Ala Ala Gly Ile Pro Asp Arg Phe Ser Gly Ser Arg 50 55 60 Trp Gly Pro Asp Tyr Asn Leu Thr Ile Ser Asn Leu Glu Ser Gly Asp 65 70 75 80 Phe Gly Val Tyr Tyr Cys Gln Gln Tyr Glu Phe Phe Gly Gln Gly Thr 85 90 95

Lys Val Gln Val Asp Ile Lys 100

<210> 29 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Variant Antibody Sequence

Page 25

Sequence_Listing <400> 29

Gln Val 1 Arg Leu Ser Gln Ser Gly Gly Gln Met Lys Lys Pro Gly 15 Asp 5 10 Ser Met Arg Ile Ser Cys Arg Ala Ser Gly Tyr Glu Phe Ile Asn Cys 20 25 30 Pro Ile Asn Trp Ile Arg Leu Ala Pro Gly Lys Arg Pro Glu Trp Met 35 40 45 Gly Trp Met Lys Pro Arg Gly Gly Ala Val Ser Tyr Ala Arg Gln Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Met Tyr Ser Glu Thr Ala Phe 65 70 75 80 Leu Glu Leu Arg Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95 Thr Arg Gly Lys Tyr Cys Thr Ala Arg Asp Tyr Tyr Asn Trp Asp Phe 100 105 110 Glu His Trp Gly Gln Gly Thr Pro Val Thr Val Ser Ser 115 120 125 <210> 30 <211> 125 <212> PRT <213> , Artificial Sequence <220> <223> Variant Antibody Sequence <400> 30 Gln Val Arg Leu Ser Gln Ser Gly Gly Gln Met Lys Lys Pro Gly Asp 1 5 10 15 Ser Met Arg Ile Ser Cys Arg Ala Ser Gly Tyr Glu Phe Ile Asn Cys 20 25 30 Pro Ile Asn Trp Ile Arg Leu Ala Pro Gly Lys Arg Pro Glu Trp Met 35 40 45 Gly Trp Met Lys Pro Arg His Gly Ala Val Ser Tyr Ala Arg Gln Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Met Tyr Ser Glu Thr Ala Phe 65 70 75 80 Leu Glu Leu Arg Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95

Page 26

Sequence_Listing

Thr Arg Gly Lys Tyr Cys Thr Ala Arg Asp Tyr Tyr Asn Trp Asp Phe 100 105 110

Glu His Trp Gly Gln Gly Thr Pro Val Thr Val Ser Ser 115 120 125 <210> 31 <211> 101 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 31

Ser 1 Leu Thr Gln Ser 5 Pro Gly Thr Leu Ser 10 Leu Ser Pro Gly Glu 15 Thr Ala Ile Ile Ser Cys Arg Thr Ser Gln Tyr Gly Ser Leu Ala Trp Tyr 20 25 30 Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Val Ile Tyr Ser Gly Ser 35 40 45 Thr Arg Ala Ala Gly Ile Pro Asp Arg Phe Ser Gly Ser Arg Trp Gly 50 55 60 Pro Asp Tyr Asn Leu Thr Ile Ser Asn Leu Glu Ser Gly Asp Phe Gly 65 70 75 80 Val Tyr Tyr Cys Gln Gln Tyr Glu Phe Phe Gly Gln Gly Thr Lys Val 85 90 95

Gln Val Asp Ile Lys 100 <210> 32 <211> 125 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 32

Gln Val Arg Leu Ser Gln Ser Gly Gly Gln Met Lys Lys Pro Gly Asp 1 5 10 15 Ser Met Arg Ile Ser Cys Arg Ala Ser Gly Tyr Glu Phe Ile Asn Cys 20 25 30 Pro Ile Asn Trp Val Arg Leu Ala Pro Gly Lys Arg Pro Glu Trp Met

35 40 45

Page 27

Sequence_Listing

Gly Trp Met 50 Lys Pro Arg His 55 Gly Ala Val Ser Tyr Ala 60 Arg Gln Leu Gln Gly Arg Val Thr Met Thr Arg Asp Met Tyr Ser Glu Thr Ala Phe 65 70 75 80 Leu Glu Leu Arg Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Lys Tyr Cys Thr Ala Arg Asp Tyr Tyr Asn Trp Asp Phe 100 105 110 Glu His Trp Gly Gln Gly Thr Pro Val Thr Val Ser Ser 115 120 125 <210> 33 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> Variant Antibody Sequence <400> 33 Ser Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Thr 1 5 10 15 Ala Ile Ile Ser Cys Arg Thr Ser Gln Tyr Gly Ser Leu Ala Trp Tyr 20 25 30 Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Val Ile Tyr Ser Gly Ser 35 40 45 Thr Arg Ala Ala Gly Ile Pro Asp Arg Phe Ser Gly Ser Arg Trp Gly 50 55 60 Pro Asp Tyr Asn Leu Thr Ile Ser Asn Leu Glu Ser Gly Asp Phe Gly 65 70 75 80 Val Tyr Tyr Cys Gln Gln Tyr Glu Phe Phe Gly Gln Gly Thr Lys Val 85 90 95

Gln Val Asp Ile Lys 100 <210> 34 <211> 28 <212> PRT <213> Human immunodeficiency virus type 1 <400> 34

Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn 1 5 10 15

Page 28

Sequence_Listing

Trp Phe Asp Ile Thr Asn Trp Leu Trp Tyr Ile Arg 20 25 <210> 35 <211> 131 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 35

Glu 1 Val Arg Leu Val 5 Glu Ser Gly Gly Gly Leu Val 10 Lys Pro Gly 15 Gly Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Asp Asn Ala 20 25 30 Trp Met Thr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Thr Gly Pro Gly Glu Gly Trp Ser Val Asp Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr 65 70 75 80 Leu Tyr Leu Glu Met Asn Asn Val Arg Thr Glu Asp Thr Gly Tyr Tyr 85 90 95 Phe Cys Ala Arg Thr Gly Lys Tyr Tyr Asp Phe Trp Trp Gly Tyr Pro 100 105 110 Pro Gly Glu Glu Tyr Phe Gln Asp Trp Gly Gln Gly Thr Leu Val Ile

115 120 125

Val Ser Ser 130 <210> 36 <211> 131 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence

<400> 36 Glu Val Arg Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Asp Asn Ala 20 25 30

Page 29

Sequence_Listing

Trp Met Thr 35 Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 40 45 Gly Arg Ile Thr Gly Pro Gly Glu Gly Trp Ser Val Asp Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr 65 70 75 80 Leu Tyr Leu Glu Met Asn Asn Val Arg Thr Glu Asp Thr Gly Tyr Tyr 85 90 95 Phe Cys Ala Arg Thr Gly Lys Tyr Tyr Asp Phe Trp Ser Gly Cys Pro 100 105 110 Pro Gly Glu Glu Tyr Phe Gln Asp Trp Gly Gln Gly Thr Leu Val Ile 115 120 125 Val Ser Ser 130 <210> 37 <211> 131 <212> PRT <213> Artificial Sequence <220> <223> Variant Antibody Sequence <400> 37 Glu Val Arg Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Asp Asn Ala 20 25 30 Trp Met Thr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Thr Gly Pro Gly Glu Gly Trp Ser Val Asp Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr 65 70 75 80 Leu Tyr Leu Glu Met Asn Asn Val Arg Thr Glu Asp Thr Gly Tyr Tyr 85 90 95 Phe Cys Ala Arg Thr Gly Lys Tyr Tyr Asp Phe Trp Trp Gly Cys Pro 100 105 110

Page 30

Sequence_Listing

Pro Gly Glu Glu Tyr Phe Gln Asp Trp Gly Gln Gly Thr Leu Val Ile 115 120 125

Val Ser Ser 130 <210> 38 <211> 131 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 38

Glu 1 Val Arg Leu Ala Glu Ser 5 Gly Gly Gly Leu 10 Val Lys Pro Gly 15 Gly Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Asp Asn Ala 20 25 30 Trp Met Thr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Thr Gly Pro Gly Glu Gly Trp Ser Val Asp Tyr Ala Ala 50 55 60 Pro Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Tyr Lys Asn Thr 65 70 75 80 Leu Tyr Leu Glu Met Asn Asn Leu Arg Thr Glu Asp Ser Gly Leu Tyr 85 90 95 Phe Cys Ala Arg Thr Gly Lys Tyr Tyr Asp Phe Trp Trp Gly Tyr Pro 100 105 110 Pro Gly Glu Glu Tyr Phe Gln Asp Trp Gly Arg Gly Thr Leu Val Ile 115 120 125

Val Ser Ser 130 <210> 39 <211> 131 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 39

Glu Val Arg Leu Ala Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Page 31

Ser Sequence_Listing Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Asp Asn Ala 20 25 30 Trp Met Thr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Thr Gly Pro Gly Glu Gly Trp Ser Val Asp Tyr Ala Ala 50 55 60 Pro Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Tyr Lys Asn Thr 65 70 75 80 Leu Tyr Leu Glu Met Asn Asn Leu Arg Thr Glu Asp Ser Gly Leu Tyr 85 90 95 Phe Cys Ala Arg Thr Gly Lys Tyr Tyr Asp Phe Trp Ser Gly Cys Pro 100 105 110 Pro Gly Glu Glu Tyr Phe Gln Asp Trp Gly Arg Gly Thr Leu Val Ile 115 120 125 Val Ser Ser

130 <210> 40 <211> 131 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 40 Glu Val 1 Arg Leu Ala Glu 5 Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 10 15 Ser Leu Arg Leu Ser Cys 20 Ser Ala Ser Gly Phe Asp Phe Asp Asn Ala 25 30 Trp Met Thr Trp Val Arg 35 Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 40 45 Gly Arg 50 Ile Thr Gly Pro Gly Glu Gly Trp Ser Val Asp Tyr Ala Ala 55 60 Pro Val 65 Glu Gly Arg Phe 70 Thr Ile Ser Arg Asp Asn Tyr Lys Asn Thr 75 80 Leu Tyr Leu Glu Met Asn 85 Asn Leu Arg Thr Glu Asp Ser Gly Leu Tyr 90 95 Phe Cys Ala Arg Thr Gly 100 Lys Tyr Tyr Asp Phe Trp Trp Gly Cys Pro 105 110

Page 32

Sequence_Listing

Pro Gly Glu 115 Glu Tyr Phe Gln Asp Trp Gly Arg 120 Gly Thr 125 Leu Val Ile Val Ser Ser 130 <210> 41 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Variant Antibody Sequence <400> 41 Ala Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Lys Gln 1 5 10 15 Thr Val Thr Ile Thr Cys Arg Gly Asp Ser Leu Arg Cys His Tyr Ala 20 25 30 Ser Trp Tyr Gln Lys Lys Pro Gly Gln Ala Pro Val Leu Leu Phe Tyr 35 40 45 Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ala Ser Gly Asn Arg Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Arg Asp Lys Ser Gly Ser Arg 85 90 95 Leu Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 <210> 42 <211> 109 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 42 Ala Ser 1 Glu Leu Thr Gln 5 Asp Pro Ala Val Ser Val Ala Leu Lys Gln 10 15 Thr Val Thr Ile Thr Cys 20 Arg Gly Asp Ser Leu Arg Ser His Trp Ala 25 30 Ser Trp Tyr Gln Lys Lys 35 Pro Gly Gln Ala Pro Val Leu Leu Phe Tyr 40 45

Page 33

Sequence_Listing

Gly Lys Asn Asn Arg 50 Pro Ser Gly Ile 55 Pro Asp Arg 60 Phe Ser Gly Ser Ala Ser Gly Asn Arg Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Arg Asp Lys Ser Gly Ser Arg 85 90 95 Leu Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 <210> 43 <211> 109 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 43 Ala Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Lys Gln 1 5 10 15 Thr Val Thr Ile Thr Cys Arg Gly Asp Ser Leu Arg Ser His Phe Ala 20 25 30 Ser Trp Tyr Gln Lys Lys Pro Gly Gln Ala Pro Val Leu Leu Phe Tyr 35 40 45 Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ala Ser Gly Asn Arg Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Arg Asp Lys Ser Gly Ser Arg 85 90 95 Leu Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 <210> 44 <211> 393 <212> DNA <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 44 gaagtgcggc tggctgagag cggcgggggg ctggtcaaac ctggcgggtc actgcggctg tcctgttctg cctccggctt cgattttgat aacgcatgga tgacatgggt gcgacagcca Page 34

120

Sequence_Listing cctggaaagg ggctggagtg ggtcggcaga atcactggac ctggcgaagg gtggtctgtg 180 gactacgcag ctccagtcga gggacgattc accattagta gagataacta caagaataca 240 ctgtatctgg agatgaacaa tctgaggact gaagacagcg gcctgtattt ctgcgcccgc 300 accgggaaat actatgattt ttggtctggg tacccacccg gagaggaata ttttcaggac 360 tggggacggg gcaccctggt gatcgtcagc tcc 393 <210> 45 <211> 393 <212> DNA <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 45 gaggttagac tggtggagtc aggagggggg cttgtgaagc ccggtgggtc tctccgcctg 60 agctgttctg cctccggctt tgatttcgat aacgcctgga tgacctgggt caggcagcct 120 ccaggtaagg gactggagtg ggtgggaaga atcacaggtc caggcgaggg ctggtccgtg 180 gactacgcgg aatctgttaa agggcggttt acaatctcaa gggacaatac caagaatacc 240 ttgtatttgg agatgaacaa cgtgagaact gaagacaccg gatattactt ctgtgccaga 300 acaggcaaat actacgactt ctggtccggc tatccccctg gcgaggaata ttttcaagac 360 tggggtcagg gaacccttgt tatcgtgtcc tcc 393 <210> 46 <211> 327 <212> DNA <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 46 tcatacgaac tgactcagga cactggcgtc tctgtggcac tggggaggac tgtgactatt 60 acttgccgag gcgactcact gcggagccac tacgcttcct ggtatcagaa gaaacccggc 120 caggcacctg tgctgctgtt ctacggaaag aacaataggc catctggcat ccccgaccgc 180 ttttctggca gtgcatcagg gaaccgagcc agtctgacca ttaccggcgc ccaggctgag 240 gacgaagccg attactattg cagctcccgg gataagagcg gctccagact gagcgtgttc 300 ggaggaggaa ctaaactgac cgtcctc 327 <210> 47 <211> 390 <212> DNA <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 47 accaccatgg gatggtcatg tatcatcctt tttctagtag caactgcaac cggttctgtg 60

Page 35

Sequence_Listing accgcatccg aactgactca ggaccctgcc attacttgcc gaggcgactc actgcggagc ggccaggcac ctgtgctgct gttctacgga cgcttttctg gcagtgcatc agggaaccga gaggacgaag ccgattacta ttgcagctcc ttcggaggag gaactaaact gaccgtcctc <210> 48 <211> 5856 <212> DNA <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 48 tcgcgcgttt cggtgatgac ggtgaaaacc cagcttgtct gtaagcggat gccgggagca ttggcgggtg tcggggctgg cttaactatg accatatgcg gtgtgaaata ccgcacagat ctattggcca ttgcatacgt tgtatccata tccaacatta ccgccatgtt gacattgatt ggggtcatta gttcatagcc catatatgga cccgcctggc tgaccgccca acgacccccg catagtaacg ccaataggga ctttccattg tgcccacttg gcagtacatc aagtgtatca tgacggtaaa tggcccgcct ggcattatgc ttggcagtac atctacgtat tagtcatcgc catcaatggg cgtggatagc ggtttgactc cgtcaatggg agtttgtttt ggcaccaaaa ctccgcccca ttgacgcaaa tgggcggtag agctcgttta gtgaaccgtc agatcgcctg tagaagacac cgggaccgat ccagcctcca cgccctacct gaggccgcca tccacgccgg ggtgcctcct gaactgcgtc cgccgtctag ctttgtccgg cgctcccttg gagcctacct accctgcttg ctcaactcta gttaacggtg gctgccgcgc gcgccaccag acataatagc ggtcttttct gcagtcaccg tcgtcgacac

gtctctgtgg cactgaagca gactgtgact 120 cactacgctt cctggtatca gaagaaaccc 180 aagaacaata ggccatctgg catccccgac 240 gccagtctga ccattaccgg cgcccaggct 300 cgggataaga gcggctccag actgagcgtg 360 390

tctgacacat gcagctcccg gagacggtca 60 gacaagcccg tcagggcgcg tcagcgggtg 120 cggcatcaga gcagattgta ctgagagtgc 180 gcgtaaggag aaaataccgc atcagattgg 240 tcataatatg tacatttata ttggctcatg 300 attgactagt tattaatagt aatcaattac 360 gttccgcgtt acataactta cggtaaatgg 420 cccattgacg tcaataatga cgtatgttcc 480 acgtcaatgg gtggagtatt tacggtaaac 540 tatgccaagt acgcccccta ttgacgtcaa 600 ccagtacatg accttatggg actttcctac 660 tattaccatg gtgatgcggt tttggcagta 720 acggggattt ccaagtctcc accccattga 780 tcaacgggac tttccaaaat gtcgtaacaa 840 gcgtgtacgg tgggaggtct atataagcag 900 gagacgccat ccacgctgtt ttgacctcca 960 tcggctcgca tctctccttc acgcgcccgc 1020 ttgagtcgcg ttctgccgcc tcccgcctgt 1080 gtaagtttaa agctcaggtc gagaccgggc 1140 agactcagcc ggctctccac gctttgcctg 1200 gagggcagtg tagtctgagc agtactcgtt 1260 tgacagacta acagactgtt cctttccatg 1320 gtgtgatcag atatcgcggc cgctctagac Page 36 1380

Sequence_Listing

caccatggga tggtcatgta tcatcctttt tctagtagca actgcaaccg gtgtacattc 1440 tgaagtgcgg ctggctgaga gcggcggggg gctggtcaaa cctggcgggt cactgcggct 1500 gtcctgttct gcctccggct tcgattttga taacgcatgg atgacatggg tgcgacagcc 1560 acctggaaag gggctggagt gggtcggcag aatcactgga cctggcgaag ggtggtctgt 1620 ggactacgca gctccagtcg agggacgatt caccattagt agagataact acaagaatac 1680 actgtatctg gagatgaaca atctgaggac tgaagacagc ggcctgtatt tctgcgcccg 1740 caccgggaaa tactatgatt tttggtctgg gtacccaccc ggagaggaat attttcagga 1800 ctggggacgg ggcaccctgg tgatcgtcag ctccgcgtcg accaagggcc catcggtctt 1860 ccccctggca ccctcctcca agagcacctc tgggggcaca gcggccctgg gctgcctggt 1920 caaggactac ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc tgaccagcgg 1980 cgtgcacacc ttcccggctg tcctacagtc ctcaggactc tactccctca gcagcgtggt 2040 gaccgtgccc tccagcagct tgggcaccca gacctacatc tgcaacgtga atcacaagcc 2100 cagcaacacc aaggtggaca agaaagttga gcccaaatct tgtgacaaaa ctcacacatg 2160 cccaccgtgc ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa 2220 acccaaggac accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt 2280 gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa 2340 tgccaagaca aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct 2400 caccgtcctg caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa 2460 agccctccca gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc 2520 acaggtgtac accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac 2580 ctgcctggtc aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca 2640 gccggagaac aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct 2700 ctacagcaag ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc 2760 cgtgatgcat gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg 2820 taaatgatga ggatccagat ctgctgtgcc ttctagttgc cagccatctg ttgtttgccc 2880 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa 2940 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg 3000 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg 3060 ctctatgggt acccaggtgc tgaagaattg acccggttcc tcctgggcca gaaagaagca 3120 ggcacatccc cttctctgtg acacaccctg tccacgcccc tggttcttag ttccagcccc 3180 actcatagga cactcatagc tcaggagggc tccgccttca atcccacccg ctaaagtact 3240 tggagcggtc tctccctccc tcatcagccc accaaaccaa acctagcctc caagagtggg 3300 aagaaattaa agcaagatag gctattaagt gcagagggag agaaaatgcc tccaacatgt 3360 gaggaagtaa tgagagaaat catagaattt taaggccatg atttaaggcc atcatggcct Page 37 3420

Sequence_Listing

taatcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 3480 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 3540 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 3600 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 3660 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 3720 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 3780 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 3840 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 3900 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 3960 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 4020 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 4080 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 4140 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 4200 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 4260 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 4320 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 4380 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcggg 4440 gggggggggc gctgaggtct gcctcgtgaa gaaggtgttg ctgactcata ccaggcctga 4500 atcgccccat catccagcca gaaagtgagg gagccacggt tgatgagagc tttgttgtag 4560 gtggaccagt tggtgatttt gaacttttgc tttgccacgg aacggtctgc gttgtcggga 4620 agatgcgtga tctgatcctt caactcagca aaagttcgat ttattcaaca aagccgccgt 4680 cccgtcaagt cagcgtaatg ctctgccagt gttacaacca attaaccaat tctgattaga 4740 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat 4800 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga 4860 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta 4920 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat 4980 ccggtgagaa tggcaaaagc ttatgcattt ctttccagac ttgttcaaca ggccagccat 5040 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct 5100 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca 5160 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt 5220 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag 5280 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc 5340 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact 5400 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat Page 38 5460

Sequence_Listing cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctcg 5520 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag 5580 cagacagttt tattgttcat gatgatatat ttttatcttg tgcaatgtaa catcagagat 5640 tttgagacac aacgtggctt tccccccccc cccattattg aagcatttat cagggttatt 5700 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 5760 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa 5820 cctataaaaa taggcgtatc acgaggccct ttcgtc 5856 <210> 49 <211> 5856 <212> DNA <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 49

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240 ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300 tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900 agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960 tagaagacac cgggaccgat ccagcctcca tcggctcgca tctctccttc acgcgcccgc 1020 cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt 1080 ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc 1140 ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg 1200 accctgcttg ctcaactcta gttaacggtg gagggcagtg tagtctgagc agtactcgtt 1260 gctgccgcgc gcgccaccag acataatagc tgacagacta acagactgtt cctttccatg Page 39 1320

Sequence_Listing

ggtcttttct gcagtcaccg tcgtcgacac gtgtgatcag atatcgcggc cgctctagac 1380 caccatggga tggtcatgta tcatcctttt tctagtagca actgcaaccg gtgtacattc 1440 tgaggttaga ctggtggagt caggaggggg gcttgtgaag cccggtgggt ctctccgcct 1500 gagctgttct gcctccggct ttgatttcga taacgcctgg atgacctggg tcaggcagcc 1560 tccaggtaag ggactggagt gggtgggaag aatcacaggt ccaggcgagg gctggtccgt 1620 ggactacgcg gaatctgtta aagggcggtt tacaatctca agggacaata ccaagaatac 1680 cttgtatttg gagatgaaca acgtgagaac tgaagacacc ggatattact tctgtgccag 1740 aacaggcaaa tactacgact tctggtccgg ctatccccct ggcgaggaat attttcaaga 1800 ctggggtcag ggaacccttg ttatcgtgtc ctccgcgtcg accaagggcc catcggtctt 1860 ccccctggca ccctcctcca agagcacctc tgggggcaca gcggccctgg gctgcctggt 1920 caaggactac ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc tgaccagcgg 1980 cgtgcacacc ttcccggctg tcctacagtc ctcaggactc tactccctca gcagcgtggt 2040 gaccgtgccc tccagcagct tgggcaccca gacctacatc tgcaacgtga atcacaagcc 2100 cagcaacacc aaggtggaca agaaagttga gcccaaatct tgtgacaaaa ctcacacatg 2160 cccaccgtgc ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa 2220 acccaaggac accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt 2280 gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa 2340 tgccaagaca aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct 2400 caccgtcctg caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa 2460 agccctccca gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc 2520 acaggtgtac accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac 2580 ctgcctggtc aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca 2640 gccggagaac aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct 2700 ctacagcaag ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc 2760 cgtgatgcat gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg 2820 taaatgatga ggatccagat ctgctgtgcc ttctagttgc cagccatctg ttgtttgccc 2880 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa 2940 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg 3000 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg 3060 ctctatgggt acccaggtgc tgaagaattg acccggttcc tcctgggcca gaaagaagca 3120 ggcacatccc cttctctgtg acacaccctg tccacgcccc tggttcttag ttccagcccc 3180 actcatagga cactcatagc tcaggagggc tccgccttca atcccacccg ctaaagtact 3240 tggagcggtc tctccctccc tcatcagccc accaaaccaa acctagcctc caagagtggg 3300 aagaaattaa agcaagatag gctattaagt gcagagggag Page 40 agaaaatgcc tccaacatgt 3360

Sequence_Listing

gaggaagtaa tgagagaaat catagaattt taaggccatg atttaaggcc atcatggcct 3420 taatcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 3480 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 3540 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 3600 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 3660 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 3720 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 3780 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 3840 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 3900 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 3960 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 4020 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 4080 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 4140 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 4200 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 4260 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 4320 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 4380 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcggg 4440 gggggggggc gctgaggtct gcctcgtgaa gaaggtgttg ctgactcata ccaggcctga 4500 atcgccccat catccagcca gaaagtgagg gagccacggt tgatgagagc tttgttgtag 4560 gtggaccagt tggtgatttt gaacttttgc tttgccacgg aacggtctgc gttgtcggga 4620 agatgcgtga tctgatcctt caactcagca aaagttcgat ttattcaaca aagccgccgt 4680 cccgtcaagt cagcgtaatg ctctgccagt gttacaacca attaaccaat tctgattaga 4740 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat 4800 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga 4860 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta 4920 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat 4980 ccggtgagaa tggcaaaagc ttatgcattt ctttccagac ttgttcaaca ggccagccat 5040 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct 5100 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca 5160 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt 5220 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag 5280 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc 5340 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact 5400

Page 41

Sequence_Listing ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat 5460 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctcg 5520 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag 5580 cagacagttt tattgttcat gatgatatat ttttatcttg tgcaatgtaa catcagagat 5640 tttgagacac aacgtggctt tccccccccc cccattattg aagcatttat cagggttatt 5700 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 5760 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa 5820 cctataaaaa taggcgtatc acgaggccct ttcgtc 5856 <210> 50 <211> 5119 <212> DNA <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 50

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240 ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300 tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900 agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960 tagaagacac cgggaccgat ccagcctcca tcggctcgca tctctccttc acgcgcccgc 1020 cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt 1080 ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc 1140 ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg 1200 accctgcttg ctcaactcta gttaacggtg gagggcagtg Page 42 tagtctgagc agtactcgtt 1260

Sequence_Listing

gctgccgcgc gcgccaccag acataatagc tgacagacta acagactgtt cctttccatg 1320 ggtcttttct gcagtcaccg tcgtcgacac gtgtgatcag atatcgcggc cgctctagac 1380 caccatggga tggtcatgta tcatcctttt tctagtagca actgcaaccg gttctgtgac 1440 ctcatacgaa ctgactcagg acactggcgt ctctgtggca ctggggagga ctgtgactat 1500 tacttgccga ggcgactcac tgcggagcca ctacgcttcc tggtatcaga agaaacccgg 1560 ccaggcacct gtgctgctgt tctacggaaa gaacaatagg ccatctggca tccccgaccg 1620 cttttctggc agtgcatcag ggaaccgagc cagtctgacc attaccggcg cccaggctga 1680 ggacgaagcc gattactatt gcagctcccg ggataagagc ggctccagac tgagcgtgtt 1740 cggaggagga actaaactga ccgtcctcag tcagcccaag gctgccccct cggtcactct 1800 gttcccgccc tcgagtgagg agcttcaagc caacaaggcc acactggtgt gtctcataag 1860 tgacttctac ccgggagccg tgacagtggc ctggaaggca gatagcagcc ccgtcaaggc 1920 gggagtggag accaccacac cctccaaaca aagcaacaac aagtacgcgg ccagcagcta 1980 cctgagcctg acgcctgagc agtggaagtc ccacagaagc tacagctgcc aggtcacgca 2040 tgaagggagc accgtggaga agacagtggc ccctacagaa tgttcataga agctgatcca 2100 gatctgctgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct 2160 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc 2220 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg 2280 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg ggtacccagg 2340 tgctgaagaa ttgacccggt tcctcctggg ccagaaagaa gcaggcacat ccccttctct 2400 gtgacacacc ctgtccacgc ccctggttct tagttccagc cccactcata ggacactcat 2460 agctcaggag ggctccgcct tcaatcccac ccgctaaagt acttggagcg gtctctccct 2520 ccctcatcag cccaccaaac caaacctagc ctccaagagt gggaagaaat taaagcaaga 2580 taggctatta agtgcagagg gagagaaaat gcctccaaca tgtgaggaag taatgagaga 2640 aatcatagaa ttttaaggcc atgatttaag gccatcatgg ccttaatctt ccgcttcctc 2700 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 2760 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 2820 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 2880 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 2940 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 3000 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 3060 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 3120 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 3180 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 3240 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag Page 43 tggtggccta actacggcta 3300

Sequence_Listing

cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 3360 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 3420 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac 3480 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc 3540 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag 3600 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc 3660 agcgatctgt ctatttcgtt catccatagt tgcctgactc gggggggggg ggcgctgagg 3720 tctgcctcgt gaagaaggtg ttgctgactc ataccaggcc tgaatcgccc catcatccag 3780 ccagaaagtg agggagccac ggttgatgag agctttgttg taggtggacc agttggtgat 3840 tttgaacttt tgctttgcca cggaacggtc tgcgttgtcg ggaagatgcg tgatctgatc 3900 cttcaactca gcaaaagttc gatttattca acaaagccgc cgtcccgtca agtcagcgta 3960 atgctctgcc agtgttacaa ccaattaacc aattctgatt agaaaaactc atcgagcatc 4020 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt 4080 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat 4140 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa 4200 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa 4260 agcttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa 4320 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg 4380 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact 4440 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct 4500 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc 4560 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta 4620 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc 4680 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac 4740 ccatataaat cagcatccat gttggaattt aatcgcggcc tcgagcaaga cgtttcccgt 4800 tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt 4860 catgatgata tatttttatc ttgtgcaatg taacatcaga gattttgaga cacaacgtgg 4920 ctttcccccc ccccccatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 4980 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 5040 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt 5100 atcacgaggc cctttcgtc 5119

<210> 51 <211> 5119 <212> DNA <213> Artificial Sequence

Page 44

Sequence_Listing <220>

<223> Variant Antibody Sequence <400> 51

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240 ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300 tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900 agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960 tagaagacac cgggaccgat ccagcctcca tcggctcgca tctctccttc acgcgcccgc 1020 cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt 1080 ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc 1140 ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg 1200 accctgcttg ctcaactcta gttaacggtg gagggcagtg tagtctgagc agtactcgtt 1260 gctgccgcgc gcgccaccag acataatagc tgacagacta acagactgtt cctttccatg 1320 ggtcttttct gcagtcaccg tcgtcgacac gtgtgatcag atatcgcggc cgctctagac 1380 caccatggga tggtcatgta tcatcctttt tctagtagca actgcaaccg gttctgtgac 1440 cgcatccgaa ctgactcagg accctgccgt ctctgtggca ctgaagcaga ctgtgactat 1500 tacttgccga ggcgactcac tgcggagcca ctacgcttcc tggtatcaga agaaacccgg 1560 ccaggcacct gtgctgctgt tctacggaaa gaacaatagg ccatctggca tccccgaccg 1620 cttttctggc agtgcatcag ggaaccgagc cagtctgacc attaccggcg cccaggctga 1680 ggacgaagcc gattactatt gcagctcccg ggataagagc ggctccagac tgagcgtgtt 1740 cggaggagga actaaactga ccgtcctcag tcagcccaag gctgccccct cggtcactct 1800 gttcccgccc tcgagtgagg agcttcaagc caacaaggcc acactggtgt gtctcataag 1860 tgacttctac ccgggagccg tgacagtggc ctggaaggca Page 45 gatagcagcc ccgtcaaggc 1920

Sequence_Listing

gggagtggag accaccacac cctccaaaca aagcaacaac aagtacgcgg ccagcagcta 1980 cctgagcctg acgcctgagc agtggaagtc ccacagaagc tacagctgcc aggtcacgca 2040 tgaagggagc accgtggaga agacagtggc ccctacagaa tgttcataga agctgatcca 2100 gatctgctgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct 2160 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc 2220 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg 2280 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg ggtacccagg 2340 tgctgaagaa ttgacccggt tcctcctggg ccagaaagaa gcaggcacat ccccttctct 2400 gtgacacacc ctgtccacgc ccctggttct tagttccagc cccactcata ggacactcat 2460 agctcaggag ggctccgcct tcaatcccac ccgctaaagt acttggagcg gtctctccct 2520 ccctcatcag cccaccaaac caaacctagc ctccaagagt gggaagaaat taaagcaaga 2580 taggctatta agtgcagagg gagagaaaat gcctccaaca tgtgaggaag taatgagaga 2640 aatcatagaa ttttaaggcc atgatttaag gccatcatgg ccttaatctt ccgcttcctc 2700 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 2760 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 2820 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 2880 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 2940 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 3000 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 3060 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 3120 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 3180 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 3240 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 3300 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 3360 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 3420 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac 3480 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc 3540 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag 3600 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc 3660 agcgatctgt ctatttcgtt catccatagt tgcctgactc gggggggggg ggcgctgagg 3720 tctgcctcgt gaagaaggtg ttgctgactc ataccaggcc tgaatcgccc catcatccag 3780 ccagaaagtg agggagccac ggttgatgag agctttgttg taggtggacc agttggtgat 3840 tttgaacttt tgctttgcca cggaacggtc tgcgttgtcg ggaagatgcg tgatctgatc 3900 cttcaactca gcaaaagttc gatttattca acaaagccgc Page 46 cgtcccgtca agtcagcgta 3960

Sequence_Listing

atgctctgcc agtgttacaa ccaattaacc aattctgatt agaaaaactc atcgagcatc 4020 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt 4080 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat 4140 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa 4200 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa 4260 agcttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa 4320 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg 4380 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact 4440 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct 4500 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc 4560 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta 4620 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc 4680 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac 4740 ccatataaat cagcatccat gttggaattt aatcgcggcc tcgagcaaga cgtttcccgt 4800 tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt 4860 catgatgata tatttttatc ttgtgcaatg taacatcaga gattttgaga cacaacgtgg 4920 ctttcccccc ccccccatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 4980 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 5040 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt 5100 atcacgaggc cctttcgtc 5119

<210> 52 <211> 22 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 52

Ala Arg Thr Gly Lys Tyr Tyr Asp Phe Trp Trp Gly Tyr Pro Pro Gly 1 5 10 15

Glu Glu Tyr Phe Gln Asp 20 <210> <211> <212> <213> 53 22 PRT Artificial Sequence <220> <223> Variant Antibody Sequence

Page 47

Sequence_Listing <400> 53

Ala Arg Thr Gly Lys Tyr Tyr Asp Phe Trp Ser Gly Cys Pro Pro Gly 15 1 5 10 Glu Glu Tyr Phe Gln Asp 20 <210> <211> <212> <213> 54 22 PRT Artificial Sequence <220> <223> Variant Antibody Sequence <400> 54

Ala Arg Thr Gly Lys Tyr Tyr Asp Phe Trp Trp Gly Cys Pro Pro Gly 1 5 10 15

Glu Glu Tyr Phe Gln Asp 20 <210> 55 <211> 6 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 55

Ser Leu Arg Cys His Tyr 1 5 <210> 56 <211> 6 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 56

Ser Leu Arg Ser His Trp 1 5 <210> 57 <211> 6 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 57

Ser Leu Arg Ser His Phe 1 5

Page 48

Sequence_Listing <210> 58 <211> 109 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 58

Ala 1 Ser Glu Leu Thr 5 Gln Asp Pro Ala Val 10 Ser Val Ala Leu Lys 15 Gln Thr Val Thr Ile Thr Cys Arg Gly Asp Ser Leu Arg Cys His Trp Ala 20 25 30 Ser Trp Tyr Gln Lys Lys Pro Gly Gln Ala Pro Val Leu Leu Phe Tyr 35 40 45 Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ala Ser Gly Asn Arg Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Arg Asp Lys Ser Gly Ser Arg 85 90 95 Leu Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 <210> 59 <211> 109 <212> PRT <213> Artificial Sequence

<220> <223> <400> Ala Ser 1 Variant Antibody Sequence 59 Val Ala Leu Lys 15 Gln Glu Leu Thr 5 Gln Asp Pro Ala Val 10 Ser Thr Val Thr Ile 20 Thr Cys Arg Gly Asp 25 Ser Leu Arg Cys His 30 Phe Ala Ser Trp Tyr 35 Gln Lys Lys Pro Gly Gln 40 Ala Pro Val Leu 45 Leu Phe Tyr Gly Lys 50 Asn Asn Arg Pro Ser 55 Gly Ile Pro Asp Arg 60 Phe Ser Gly Ser Ala Ser 65 Gly Asn Arg Ala 70 Ser Leu Thr Ile Thr 75 Page 49 Gly Ala Gln Ala Glu 80

Sequence_Listing

Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Arg Asp Lys Ser Gly Ser Arg 85 90 95

Leu Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 60 <211> 5856 <212> DNA <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 60

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240 ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300 tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900 agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960 tagaagacac cgggaccgat ccagcctcca tcggctcgca tctctccttc acgcgcccgc 1020 cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt 1080 ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc 1140 ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg 1200 accctgcttg ctcaactcta gttaacggtg gagggcagtg tagtctgagc agtactcgtt 1260 gctgccgcgc gcgccaccag acataatagc tgacagacta acagactgtt cctttccatg 1320 ggtcttttct gcagtcaccg tcgtcgacac gtgtgatcag atatcgcggc cgctctagac 1380 caccatggga tggtcatgta tcatcctttt tctagtagca actgcaaccg gtgtacattc 1440 tgaggttaga ctggtggagt caggaggggg gcttgtgaag cccggtgggt ctctccgcct Page 50 1500

Sequence_Listing

gagctgttct gcctccggct ttgatttcga taacgcctgg atgacctggg tcaggcagcc 1560 tccaggtaag ggactggagt gggtgggaag aatcacaggt ccaggcgagg gctggtccgt 1620 ggactacgcg gaatctgtta aagggcggtt tacaatctca agggacaata ccaagaatac 1680 cttgtatttg gagatgaaca acgtgagaac tgaagacacc ggatattact tctgtgccag 1740 aacaggcaaa tactacgact tctggtccgg ctgcccccct ggcgaggaat attttcaaga 1800 ctggggtcag ggaacccttg ttatcgtgtc ctccgcgtcg accaagggcc catcggtctt 1860 ccccctggca ccctcctcca agagcacctc tgggggcaca gcggccctgg gctgcctggt 1920 caaggactac ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc tgaccagcgg 1980 cgtgcacacc ttcccggctg tcctacagtc ctcaggactc tactccctca gcagcgtggt 2040 gaccgtgccc tccagcagct tgggcaccca gacctacatc tgcaacgtga atcacaagcc 2100 cagcaacacc aaggtggaca agaaagttga gcccaaatct tgtgacaaaa ctcacacatg 2160 cccaccgtgc ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa 2220 acccaaggac accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt 2280 gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa 2340 tgccaagaca aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct 2400 caccgtcctg caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa 2460 agccctccca gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc 2520 acaggtgtac accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac 2580 ctgcctggtc aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca 2640 gccggagaac aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct 2700 ctacagcaag ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc 2760 cgtgatgcat gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg 2820 taaatgatga ggatccagat ctgctgtgcc ttctagttgc cagccatctg ttgtttgccc 2880 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa 2940 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg 3000 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg 3060 ctctatgggt acccaggtgc tgaagaattg acccggttcc tcctgggcca gaaagaagca 3120 ggcacatccc cttctctgtg acacaccctg tccacgcccc tggttcttag ttccagcccc 3180 actcatagga cactcatagc tcaggagggc tccgccttca atcccacccg ctaaagtact 3240 tggagcggtc tctccctccc tcatcagccc accaaaccaa acctagcctc caagagtggg 3300 aagaaattaa agcaagatag gctattaagt gcagagggag agaaaatgcc tccaacatgt 3360 gaggaagtaa tgagagaaat catagaattt taaggccatg atttaaggcc atcatggcct 3420 taatcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 3480 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 3540

Page 51

Sequence_Listing

aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 3600 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 3660 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 3720 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 3780 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 3840 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 3900 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 3960 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 4020 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 4080 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 4140 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 4200 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 4260 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 4320 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 4380 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcggg 4440 gggggggggc gctgaggtct gcctcgtgaa gaaggtgttg ctgactcata ccaggcctga 4500 atcgccccat catccagcca gaaagtgagg gagccacggt tgatgagagc tttgttgtag 4560 gtggaccagt tggtgatttt gaacttttgc tttgccacgg aacggtctgc gttgtcggga 4620 agatgcgtga tctgatcctt caactcagca aaagttcgat ttattcaaca aagccgccgt 4680 cccgtcaagt cagcgtaatg ctctgccagt gttacaacca attaaccaat tctgattaga 4740 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat 4800 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga 4860 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta 4920 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat 4980 ccggtgagaa tggcaaaagc ttatgcattt ctttccagac ttgttcaaca ggccagccat 5040 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct 5100 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca 5160 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt 5220 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag 5280 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc 5340 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact 5400 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat 5460 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctcg 5520 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag Page 52 5580

Sequence_Listing cagacagttt tattgttcat gatgatatat ttttatcttg tgcaatgtaa catcagagat 5640 tttgagacac aacgtggctt tccccccccc cccattattg aagcatttat cagggttatt 5700 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 5760 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa 5820 cctataaaaa taggcgtatc acgaggccct ttcgtc 5856 <210> 61 <211> 5119

<212> DNA <213> Artificial Sequence <220> <223> Variant Antibody Sequence <400> 61 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240 ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300 tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900 agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960 tagaagacac cgggaccgat ccagcctcca tcggctcgca tctctccttc acgcgcccgc 1020 cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt 1080 ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc 1140 ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg 1200 accctgcttg ctcaactcta gttaacggtg gagggcagtg tagtctgagc agtactcgtt 1260 gctgccgcgc gcgccaccag acataatagc tgacagacta acagactgtt cctttccatg 1320 ggtcttttct gcagtcaccg tcgtcgacac gtgtgatcag atatcgcggc cgctctagac 1380 caccatggga tggtcatgta tcatcctttt tctagtagca Page 53 actgcaaccg gttctgtgac 1440

Sequence_Listing

cgcatccgaa ctgactcagg accctgccgt ctctgtggca ctgaagcaga ctgtgactat 1500 tacttgccga ggcgactcac tgcggtgcca ctacgcttcc tggtatcaga agaaacccgg 1560 ccaggcacct gtgctgctgt tctacggaaa gaacaatagg ccatctggca tccccgaccg 1620 cttttctggc agtgcatcag ggaaccgagc cagtctgacc attaccggcg cccaggctga 1680 ggacgaagcc gattactatt gcagctcccg ggataagagc ggctccagac tgagcgtgtt 1740 cggaggagga actaaactga ccgtcctcag tcagcccaag gctgccccct cggtcactct 1800 gttcccgccc tcgagtgagg agcttcaagc caacaaggcc acactggtgt gtctcataag 1860 tgacttctac ccgggagccg tgacagtggc ctggaaggca gatagcagcc ccgtcaaggc 1920 gggagtggag accaccacac cctccaaaca aagcaacaac aagtacgcgg ccagcagcta 1980 cctgagcctg acgcctgagc agtggaagtc ccacagaagc tacagctgcc aggtcacgca 2040 tgaagggagc accgtggaga agacagtggc ccctacagaa tgttcataga agctgatcca 2100 gatctgctgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct 2160 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc 2220 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg 2280 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg ggtacccagg 2340 tgctgaagaa ttgacccggt tcctcctggg ccagaaagaa gcaggcacat ccccttctct 2400 gtgacacacc ctgtccacgc ccctggttct tagttccagc cccactcata ggacactcat 2460 agctcaggag ggctccgcct tcaatcccac ccgctaaagt acttggagcg gtctctccct 2520 ccctcatcag cccaccaaac caaacctagc ctccaagagt gggaagaaat taaagcaaga 2580 taggctatta agtgcagagg gagagaaaat gcctccaaca tgtgaggaag taatgagaga 2640 aatcatagaa ttttaaggcc atgatttaag gccatcatgg ccttaatctt ccgcttcctc 2700 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 2760 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 2820 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 2880 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 2940 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 3000 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 3060 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 3120 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 3180 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 3240 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 3300 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 3360 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 3420 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac Page 54 3480

Sequence_Listing

ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc 3540 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag 3600 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc 3660 agcgatctgt ctatttcgtt catccatagt tgcctgactc gggggggggg ggcgctgagg 3720 tctgcctcgt gaagaaggtg ttgctgactc ataccaggcc tgaatcgccc catcatccag 3780 ccagaaagtg agggagccac ggttgatgag agctttgttg taggtggacc agttggtgat 3840 tttgaacttt tgctttgcca cggaacggtc tgcgttgtcg ggaagatgcg tgatctgatc 3900 cttcaactca gcaaaagttc gatttattca acaaagccgc cgtcccgtca agtcagcgta 3960 atgctctgcc agtgttacaa ccaattaacc aattctgatt agaaaaactc atcgagcatc 4020 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt 4080 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat 4140 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa 4200 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa 4260 agcttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa 4320 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg 4380 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact 4440 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct 4500 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc 4560 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta 4620 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc 4680 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac 4740 ccatataaat cagcatccat gttggaattt aatcgcggcc tcgagcaaga cgtttcccgt 4800 tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt 4860 catgatgata tatttttatc ttgtgcaatg taacatcaga gattttgaga cacaacgtgg 4920 ctttcccccc ccccccatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 4980 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 5040 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt 5100 atcacgaggc cctttcgtc 5119

<210> 62 <211> 5856 <212> DNA <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 62 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

Page 55

Sequence_Listing

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240 ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300 tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900 agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960 tagaagacac cgggaccgat ccagcctcca tcggctcgca tctctccttc acgcgcccgc 1020 cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt 1080 ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc 1140 ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg 1200 accctgcttg ctcaactcta gttaacggtg gagggcagtg tagtctgagc agtactcgtt 1260 gctgccgcgc gcgccaccag acataatagc tgacagacta acagactgtt cctttccatg 1320 ggtcttttct gcagtcaccg tcgtcgacac gtgtgatcag atatcgcggc cgctctagac 1380 caccatgggc tggtcctgta ttatcctgtt cctggtcgca actgctactg gcgtccattc 1440 agaagtgagg ctggtcgaga gcggcggcgg cctggtgaag ccaggaggaa gcctgcgact 1500 gagctgctcc gcctctggct tcgactttga taacgcttgg atgacatggg tgcgacagcc 1560 ccctggaaaa ggcctggagt gggtcggaag aatcaccggc cccggagagg gatggagtgt 1620 ggactacgca gaatcagtca agggccggtt caccattagc cgggataaca ccaaaaatac 1680 actgtatctg gagatgaaca atgtcaggac tgaagacacc gggtactatt tctgtgcccg 1740 caccggaaag tactatgatt tttggtgggg ctacccaccc ggagaagaat actttcagga 1800 ctggggacag ggaacactgg tcatcgtcag cagcgcctcg accaagggcc catcggtctt 1860 ccccctggca ccctcctcca agagcacctc tgggggcaca gcggccctgg gctgcctggt 1920 caaggactac ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc tgaccagcgg 1980 cgtgcacacc ttcccggctg tcctacagtc ctcaggactc tactccctca gcagcgtggt 2040 gaccgtgccc tccagcagct tgggcaccca gacctacatc tgcaacgtga atcacaagcc Page 56 2100

Sequence_Listing

cagcaacacc aaggtggaca agaaagttga gcccaaatct tgtgacaaaa ctcacacatg 2160 cccaccgtgc ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa 2220 acccaaggac accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt 2280 gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa 2340 tgccaagaca aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct 2400 caccgtcctg caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa 2460 agccctccca gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc 2520 acaggtgtac accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac 2580 ctgcctggtc aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca 2640 gccggagaac aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct 2700 ctacagcaag ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc 2760 cgtgatgcat gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg 2820 taaatgatga ggatccagat ctgctgtgcc ttctagttgc cagccatctg ttgtttgccc 2880 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa 2940 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg 3000 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg 3060 ctctatgggt acccaggtgc tgaagaattg acccggttcc tcctgggcca gaaagaagca 3120 ggcacatccc cttctctgtg acacaccctg tccacgcccc tggttcttag ttccagcccc 3180 actcatagga cactcatagc tcaggagggc tccgccttca atcccacccg ctaaagtact 3240 tggagcggtc tctccctccc tcatcagccc accaaaccaa acctagcctc caagagtggg 3300 aagaaattaa agcaagatag gctattaagt gcagagggag agaaaatgcc tccaacatgt 3360 gaggaagtaa tgagagaaat catagaattt taaggccatg atttaaggcc atcatggcct 3420 taatcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 3480 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 3540 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 3600 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 3660 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 3720 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 3780 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 3840 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 3900 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 3960 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 4020 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 4080 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc Page 57 4140

Sequence_Listing

ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 4200 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 4260 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 4320 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 4380 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcggg 4440 gggggggggc gctgaggtct gcctcgtgaa gaaggtgttg ctgactcata ccaggcctga 4500 atcgccccat catccagcca gaaagtgagg gagccacggt tgatgagagc tttgttgtag 4560 gtggaccagt tggtgatttt gaacttttgc tttgccacgg aacggtctgc gttgtcggga 4620 agatgcgtga tctgatcctt caactcagca aaagttcgat ttattcaaca aagccgccgt 4680 cccgtcaagt cagcgtaatg ctctgccagt gttacaacca attaaccaat tctgattaga 4740 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat 4800 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga 4860 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta 4920 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat 4980 ccggtgagaa tggcaaaagc ttatgcattt ctttccagac ttgttcaaca ggccagccat 5040 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct 5100 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca 5160 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt 5220 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag 5280 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc 5340 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact 5400 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat 5460 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctcg 5520 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag 5580 cagacagttt tattgttcat gatgatatat ttttatcttg tgcaatgtaa catcagagat 5640 tttgagacac aacgtggctt tccccccccc cccattattg aagcatttat cagggttatt 5700 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 5760 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa 5820 cctataaaaa taggcgtatc acgaggccct ttcgtc 5856

<210> 63 <211> 5856 <212> DNA <213> Artificial Sequence <220>

<223> Variant Antibody Sequence

Page 58

Sequence_Listing

<400> 63 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240 ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300 tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900 agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960 tagaagacac cgggaccgat ccagcctcca tcggctcgca tctctccttc acgcgcccgc 1020 cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt 1080 ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc 1140 ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg 1200 accctgcttg ctcaactcta gttaacggtg gagggcagtg tagtctgagc agtactcgtt 1260 gctgccgcgc gcgccaccag acataatagc tgacagacta acagactgtt cctttccatg 1320 ggtcttttct gcagtcaccg tcgtcgacac gtgtgatcag atatcgcggc cgctctagac 1380 caccatggga tggtcatgta tcatcctttt tctagtagca actgcaaccg gtgtacattc 1440 tgaggttaga ctggtggagt caggaggggg gcttgtgaag cccggtgggt ctctccgcct 1500 gagctgttct gcctccggct ttgatttcga taacgcctgg atgacctggg tcaggcagcc 1560 tccaggtaag ggactggagt gggtgggaag aatcacaggt ccaggcgagg gctggtccgt 1620 ggactacgcg gaatctgtta aagggcggtt tacaatctca agggacaata ccaagaatac 1680 cttgtatttg gagatgaaca acgtgagaac tgaagacacc ggatattact tctgtgccag 1740 aacaggcaaa tactacgact tctggtgggg ctgcccccct ggcgaggaat attttcaaga 1800 ctggggtcag ggaacccttg ttatcgtgtc ctccgcgtcg accaagggcc catcggtctt 1860 ccccctggca ccctcctcca agagcacctc tgggggcaca gcggccctgg gctgcctggt 1920 caaggactac ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc tgaccagcgg 1980 cgtgcacacc ttcccggctg tcctacagtc ctcaggactc Page 59 tactccctca gcagcgtggt 2040

Sequence_Listing

gaccgtgccc tccagcagct tgggcaccca gacctacatc tgcaacgtga atcacaagcc 2100 cagcaacacc aaggtggaca agaaagttga gcccaaatct tgtgacaaaa ctcacacatg 2160 cccaccgtgc ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa 2220 acccaaggac accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt 2280 gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa 2340 tgccaagaca aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct 2400 caccgtcctg caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa 2460 agccctccca gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc 2520 acaggtgtac accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac 2580 ctgcctggtc aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca 2640 gccggagaac aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct 2700 ctacagcaag ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc 2760 cgtgatgcat gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg 2820 taaatgatga ggatccagat ctgctgtgcc ttctagttgc cagccatctg ttgtttgccc 2880 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa 2940 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg 3000 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg 3060 ctctatgggt acccaggtgc tgaagaattg acccggttcc tcctgggcca gaaagaagca 3120 ggcacatccc cttctctgtg acacaccctg tccacgcccc tggttcttag ttccagcccc 3180 actcatagga cactcatagc tcaggagggc tccgccttca atcccacccg ctaaagtact 3240 tggagcggtc tctccctccc tcatcagccc accaaaccaa acctagcctc caagagtggg 3300 aagaaattaa agcaagatag gctattaagt gcagagggag agaaaatgcc tccaacatgt 3360 gaggaagtaa tgagagaaat catagaattt taaggccatg atttaaggcc atcatggcct 3420 taatcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 3480 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 3540 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 3600 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 3660 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 3720 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 3780 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 3840 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 3900 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 3960 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 4020 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca Page 60 4080

Sequence_Listing

gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 4140 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 4200 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 4260 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 4320 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 4380 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcggg 4440 gggggggggc gctgaggtct gcctcgtgaa gaaggtgttg ctgactcata ccaggcctga 4500 atcgccccat catccagcca gaaagtgagg gagccacggt tgatgagagc tttgttgtag 4560 gtggaccagt tggtgatttt gaacttttgc tttgccacgg aacggtctgc gttgtcggga 4620 agatgcgtga tctgatcctt caactcagca aaagttcgat ttattcaaca aagccgccgt 4680 cccgtcaagt cagcgtaatg ctctgccagt gttacaacca attaaccaat tctgattaga 4740 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat 4800 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga 4860 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta 4920 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat 4980 ccggtgagaa tggcaaaagc ttatgcattt ctttccagac ttgttcaaca ggccagccat 5040 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct 5100 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca 5160 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt 5220 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag 5280 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc 5340 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact 5400 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat 5460 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctcg 5520 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag 5580 cagacagttt tattgttcat gatgatatat ttttatcttg tgcaatgtaa catcagagat 5640 tttgagacac aacgtggctt tccccccccc cccattattg aagcatttat cagggttatt 5700 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 5760 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa 5820 cctataaaaa taggcgtatc acgaggccct ttcgtc 5856

<210> 64 <211> 450 <212> DNA <213> Artificial Sequence <220>

Page 61

Sequence_Listing <223> Variant Antibody Sequence <400> 64 atgggctggt cctgtattat cctgttcctg gtcgcaactg ctactggcgt ccattcagaa 60 gtgaggctgg tcgagagcgg cggcggcctg gtgaagccag gaggaagcct gcgactgagc 120 tgctccgcct ctggcttcga ctttgataac gcttggatga catgggtgcg acagccccct 180 ggaaaaggcc tggagtgggt cggaagaatc accggccccg gagagggatg gagtgtggac 240 tacgcagaat cagtcaaggg ccggttcacc attagccggg ataacaccaa aaatacactg 300 tatctggaga tgaacaatgt caggactgaa gacaccgggt actatttctg tgcccgcacc 360 ggaaagtact atgatttttg gtggggctac ccacccggag aagaatactt tcaggactgg 420 ggacagggaa cactggtcat cgtcagcagc 450 <210> 65 <211> 450 <212> DNA <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 65 atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattctgag 60 gttagactgg tggagtcagg aggggggctt gtgaagcccg gtgggtctct ccgcctgagc 120 tgttctgcct ccggctttga tttcgataac gcctggatga cctgggtcag gcagcctcca 180 ggtaagggac tggagtgggt gggaagaatc acaggtccag gcgagggctg gtccgtggac 240 tacgcggaat ctgttaaagg gcggtttaca atctcaaggg acaataccaa gaataccttg 300 tatttggaga tgaacaacgt gagaactgaa gacaccggat attacttctg tgccagaaca 360 ggcaaatact acgacttctg gtccggctgc ccccctggcg aggaatattt tcaagactgg 420 ggtcagggaa cccttgttat cgtgtcctcc 450 <210> 66 <211> 450 <212> DNA <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 66 atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattctgag 60 gttagactgg tggagtcagg aggggggctt gtgaagcccg gtgggtctct ccgcctgagc 120 tgttctgcct ccggctttga tttcgataac gcctggatga cctgggtcag gcagcctcca 180 ggtaagggac tggagtgggt gggaagaatc acaggtccag gcgagggctg gtccgtggac 240 tacgcggaat ctgttaaagg gcggtttaca atctcaaggg acaataccaa gaataccttg 300 tatttggaga tgaacaacgt gagaactgaa gacaccggat attacttctg tgccagaaca 360 ggcaaatact acgacttctg gtggggctgc ccccctggcg aggaatattt tcaagactgg 420

Page 62

Sequence_Listing ggtcagggaa cccttgttat cgtgtcctcc 450 <210> 67 <211> 393 <212> DNA <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 67 gaagtgcggc tggctgagag cggcgggggg ctggtcaaac ctggcgggtc actgcggctg 60 tcctgttctg cctccggctt cgattttgat aacgcatgga tgacatgggt gcgacagcca 120 cctggaaagg ggctggagtg ggtcggcaga atcactggac ctggcgaagg gtggtctgtg 180 gactacgcag ctccagtcga gggacgattc accattagta gagataacta caagaataca 240 ctgtatctgg agatgaacaa tctgaggact gaagacagcg gcctgtattt ctgcgcccgc 300 accgggaaat actatgattt ttggtggggg tacccacccg gagaggaata ttttcaggac 360 tggggacggg gcaccctggt gatcgtcagc tcc 393 <210> 68 <211> 393 <212> DNA <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 68 gaagtgcggc tggctgagag cggcgggggg ctggtcaaac ctggcgggtc actgcggctg 60 tcctgttctg cctccggctt cgattttgat aacgcatgga tgacatgggt gcgacagcca 120 cctggaaagg ggctggagtg ggtcggcaga atcactggac ctggcgaagg gtggtctgtg 180 gactacgcag ctccagtcga gggacgattc accattagta gagataacta caagaataca 240 ctgtatctgg agatgaacaa tctgaggact gaagacagcg gcctgtattt ctgcgcccgc 300 accgggaaat actatgattt ttggtctggg tgcccacccg gagaggaata ttttcaggac 360 tggggacggg gcaccctggt gatcgtcagc tcc 393 <210> 69 <211> 393 <212> DNA <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 69 gaagtgcggc tggctgagag cggcgggggg ctggtcaaac ctggcgggtc actgcggctg 60 tcctgttctg cctccggctt cgattttgat aacgcatgga tgacatgggt gcgacagcca 120 cctggaaagg ggctggagtg ggtcggcaga atcactggac ctggcgaagg gtggtctgtg 180 gactacgcag ctccagtcga gggacgattc accattagta gagataacta caagaataca 240

Page 63

Sequence_Listing ctgtatctgg agatgaacaa tctgaggact gaagacagcg gcctgtattt ctgcgcccgc 300 accgggaaat actatgattt ttggtggggg tgcccacccg gagaggaata ttttcaggac 360 tggggacggg gcaccctggt gatcgtcagc tcc 393 <210> 70 <211> 384 <212> DNA <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 70 atgggatggt catgtatcat cctttttcta gtagcaactg caaccggttc tgtgaccgca 60 tccgaactga ctcaggaccc tgccgtctct gtggcactga agcagactgt gactattact 120 tgccgaggcg actcactgcg gtgccactac gcttcctggt atcagaagaa acccggccag 180 gcacctgtgc tgctgttcta cggaaagaac aataggccat ctggcatccc cgaccgcttt 240 tctggcagtg catcagggaa ccgagccagt ctgaccatta ccggcgccca ggctgaggac 300 gaagccgatt actattgcag ctcccgggat aagagcggct ccagactgag cgtgttcgga 360 ggaggaacta aactgaccgt cctc 384 <210> 71 <211> 384 <212> DNA <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 71 atgggatggt catgtatcat cctttttcta gtagcaactg caaccggttc tgtgaccgca 60 tccgaactga ctcaggaccc tgccgtctct gtggcactga agcagactgt gactattact 120 tgccgaggcg actcactgcg gagccactgg gcttcctggt atcagaagaa acccggccag 180 gcacctgtgc tgctgttcta cggaaagaac aataggccat ctggcatccc cgaccgcttt 240 tctggcagtg catcagggaa ccgagccagt ctgaccatta ccggcgccca ggctgaggac 300 gaagccgatt actattgcag ctcccgggat aagagcggct ccagactgag cgtgttcgga 360 ggaggaacta aactgaccgt cctc 384 <210> 72 <211> 384 <212> DNA <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 72 atgggatggt catgtatcat cctttttcta gtagcaactg caaccggttc tgtgaccgca 60 tccgaactga ctcaggaccc tgccgtctct gtggcactga agcagactgt gactattact 120

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Sequence_Listing tgccgaggcg actcactgcg gagccacttt gcttcctggt atcagaagaa acccggccag 180 gcacctgtgc tgctgttcta cggaaagaac aataggccat ctggcatccc cgaccgcttt 240 tctggcagtg catcagggaa ccgagccagt ctgaccatta ccggcgccca ggctgaggac 300 gaagccgatt actattgcag ctcccgggat aagagcggct ccagactgag cgtgttcgga 360 ggaggaacta aactgaccgt cctc 384 <210> 73 <211> 384 <212> DNA <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 73 atgggatggt catgtatcat cctttttcta gtagcaactg caaccggttc tgtgaccgca 60 tccgaactga ctcaggaccc tgccgtctct gtggcactga agcagactgt gactattact 120 tgccgaggcg actcactgcg gagccactgg gcttcctggt atcagaagaa acccggccag 180 gcacctgtgc tgctgttcta cggaaagaac aataggccat ctggcatccc cgaccgcttt 240 tctggcagtg catcagggaa ccgagccagt ctgaccatta ccggcgccca ggctgaggac 300 gaagccgatt actattgcag ctcccgggat aagagcggct ccagactgag cgtgttcgga 360 ggaggaacta aactgaccgt cctc 384 <210> 74 <211> 384 <212> DNA <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 74 atgggatggt catgtatcat cctttttcta gtagcaactg caaccggttc tgtgaccgca 60 tccgaactga ctcaggaccc tgccgtctct gtggcactga agcagactgt gactattact 120 tgccgaggcg actcactgcg gagccacttt gcttcctggt atcagaagaa acccggccag 180 gcacctgtgc tgctgttcta cggaaagaac aataggccat ctggcatccc cgaccgcttt 240 tctggcagtg catcagggaa ccgagccagt ctgaccatta ccggcgccca ggctgaggac 300 gaagccgatt actattgcag ctcccgggat aagagcggct ccagactgag cgtgttcgga 360 ggaggaacta aactgaccgt cctc 384 <210> 75 <211> 131 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence

Page 65

Sequence_Listing <400> 75

Glu Val 1 Arg Leu Val 5 Glu Ser Gly Gly Gly 10 Leu Val Lys Pro Gly Gly 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Asp Asn Ala 20 25 30 Trp Met Thr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Thr Gly Pro Gly Glu Gly Trp Ser Val Asp Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr 65 70 75 80 Leu Tyr Leu Glu Met Asn Asn Val Arg Thr Glu Asp Thr Gly Tyr Tyr 85 90 95 Phe Cys Ala Arg Thr Gly Lys Tyr Tyr Asp Phe Trp Phe Gly Tyr Pro 100 105 110 Pro Gly Glu Glu Tyr Phe Gln Asp Trp Gly Gln Gly Thr Leu Val Ile 115 120 125 Val Ser Ser 130 <210> 76 <211> 131 <212> PRT <213> Artificial Sequence <220> <223> Variant Antibody Sequence <400> 76 Glu Val Arg Leu Arg Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Asp Asn Ala 20 25 30 Trp Met Thr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Thr Gly Pro Gly Glu Gly Trp Ser Val Asp Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr 65 70 75 80

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Leu Tyr Leu Glu Met 85 Asn Asn Val Sequence_Li sting Arg Thr 90 Glu Asp Thr Gly Tyr 95 Tyr Phe Cys Ala Arg 100 Thr Gly Lys Tyr Tyr 105 Asp Phe Trp Phe Gly 110 Tyr Pro Pro Gly Glu 115 Glu Tyr Phe Gln Asp 120 Trp Gly Gln Gly Thr 125 Leu Val Ile

Val Ser Ser 130 <210> 77 <211> 22 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 77

Ala Arg Thr Gly Lys Tyr Tyr Asp Phe Trp Phe Gly Tyr Pro Pro Gly 1 5 10 15

Glu Glu Tyr Phe Gln Asp 20 <210> 78 <211> 393 <212> DNA <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 78 gaggttagac tggtggagtc aggagggggg cttgtgaagc ccggtgggtc tctccgcctg 60 agctgttctg cctccggctt tgatttcgat aacgcctgga tgacctgggt caggcagcct 120 ccaggtaagg gactggagtg ggtgggaaga atcacaggtc caggcgaggg ctggtccgtg 180 gactacgcgg aatctgttaa agggcggttt acaatctcaa gggacaatac caagaatacc 240 ttgtatttgg agatgaacaa cgtgagaact gaagacaccg gatattactt ctgtgccaga 300 acaggcaaat actacgactt ctggttcggc tatccccctg gcgaggaata ttttcaagac 360 tggggtcagg gaacccttgt tatcgtgtcc tcc 393 <210> 79 <211> 393 <212> DNA <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 79 gaggttagac tgcgggagtc aggagggggg cttgtgaagc ccggtgggtc tctccgcctg 60

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Sequence_Listing agctgttctg cctccggctt tgatttcgat aacgcctgga tgacctgggt caggcagcct 120 ccaggtaagg gactggagtg ggtgggaaga atcacaggtc caggcgaggg ctggtccgtg 180 gactacgcgg aatctgttaa agggcggttt acaatctcaa gggacaatac caagaatacc 240 ttgtatttgg agatgaacaa cgtgagaact gaagacaccg gatattactt ctgtgccaga 300 acaggcaaat actacgactt ctggttcggc tatccccctg gcgaggaata ttttcaagac 360 tggggtcagg gaacccttgt tatcgtgtcc tcc 393 <210> 80 <211> 5856 <212> DNA <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 80

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240 ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300 tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900 agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960 tagaagacac cgggaccgat ccagcctcca tcggctcgca tctctccttc acgcgcccgc 1020 cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt 1080 ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc 1140 ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg 1200 accctgcttg ctcaactcta gttaacggtg gagggcagtg tagtctgagc agtactcgtt 1260 gctgccgcgc gcgccaccag acataatagc tgacagacta acagactgtt cctttccatg 1320 ggtcttttct gcagtcaccg tcgtcgacac gtgtgatcag atatcgcggc cgctctagac Page 68 1380

Sequence_Listing

caccatggga tggtcatgta tcatcctttt tctagtagca actgcaaccg gtgtacattc 1440 tgaggttaga ctggtggagt caggaggggg gcttgtgaag cccggtgggt ctctccgcct 1500 gagctgttct gcctccggct ttgatttcga taacgcctgg atgacctggg tcaggcagcc 1560 tccaggtaag ggactggagt gggtgggaag aatcacaggt ccaggcgagg gctggtccgt 1620 ggactacgcg gaatctgtta aagggcggtt tacaatctca agggacaata ccaagaatac 1680 cttgtatttg gagatgaaca acgtgagaac tgaagacacc ggatattact tctgtgccag 1740 aacaggcaaa tactacgact tctggttcgg ctatccccct ggcgaggaat attttcaaga 1800 ctggggtcag ggaacccttg ttatcgtgtc ctccgcgtcg accaagggcc catcggtctt 1860 ccccctggca ccctcctcca agagcacctc tgggggcaca gcggccctgg gctgcctggt 1920 caaggactac ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc tgaccagcgg 1980 cgtgcacacc ttcccggctg tcctacagtc ctcaggactc tactccctca gcagcgtggt 2040 gaccgtgccc tccagcagct tgggcaccca gacctacatc tgcaacgtga atcacaagcc 2100 cagcaacacc aaggtggaca agaaagttga gcccaaatct tgtgacaaaa ctcacacatg 2160 cccaccgtgc ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa 2220 acccaaggac accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt 2280 gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa 2340 tgccaagaca aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct 2400 caccgtcctg caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa 2460 agccctccca gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc 2520 acaggtgtac accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac 2580 ctgcctggtc aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca 2640 gccggagaac aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct 2700 ctacagcaag ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc 2760 cgtgatgcat gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg 2820 taaatgatga ggatccagat ctgctgtgcc ttctagttgc cagccatctg ttgtttgccc 2880 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa 2940 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg 3000 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg 3060 ctctatgggt acccaggtgc tgaagaattg acccggttcc tcctgggcca gaaagaagca 3120 ggcacatccc cttctctgtg acacaccctg tccacgcccc tggttcttag ttccagcccc 3180 actcatagga cactcatagc tcaggagggc tccgccttca atcccacccg ctaaagtact 3240 tggagcggtc tctccctccc tcatcagccc accaaaccaa acctagcctc caagagtggg 3300 aagaaattaa agcaagatag gctattaagt gcagagggag agaaaatgcc tccaacatgt 3360 gaggaagtaa tgagagaaat catagaattt taaggccatg atttaaggcc atcatggcct Page 69 3420

Sequence_Listing

taatcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 3480 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 3540 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 3600 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 3660 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 3720 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 3780 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 3840 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 3900 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 3960 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 4020 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 4080 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 4140 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 4200 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 4260 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 4320 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 4380 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcggg 4440 gggggggggc gctgaggtct gcctcgtgaa gaaggtgttg ctgactcata ccaggcctga 4500 atcgccccat catccagcca gaaagtgagg gagccacggt tgatgagagc tttgttgtag 4560 gtggaccagt tggtgatttt gaacttttgc tttgccacgg aacggtctgc gttgtcggga 4620 agatgcgtga tctgatcctt caactcagca aaagttcgat ttattcaaca aagccgccgt 4680 cccgtcaagt cagcgtaatg ctctgccagt gttacaacca attaaccaat tctgattaga 4740 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat 4800 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga 4860 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta 4920 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat 4980 ccggtgagaa tggcaaaagc ttatgcattt ctttccagac ttgttcaaca ggccagccat 5040 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct 5100 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca 5160 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt 5220 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag 5280 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc 5340 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact 5400 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat Page 70 5460

Sequence_Listing cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctcg 5520 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag 5580 cagacagttt tattgttcat gatgatatat ttttatcttg tgcaatgtaa catcagagat 5640 tttgagacac aacgtggctt tccccccccc cccattattg aagcatttat cagggttatt 5700 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 5760 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa 5820 cctataaaaa taggcgtatc acgaggccct ttcgtc 5856 <210> 81 <211> 5856 <212> DNA <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 81

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240 ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300 tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900 agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960 tagaagacac cgggaccgat ccagcctcca tcggctcgca tctctccttc acgcgcccgc 1020 cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt 1080 ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc 1140 ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg 1200 accctgcttg ctcaactcta gttaacggtg gagggcagtg tagtctgagc agtactcgtt 1260 gctgccgcgc gcgccaccag acataatagc tgacagacta acagactgtt cctttccatg 1320

Page 71

Sequence_Listing

ggtcttttct gcagtcaccg tcgtcgacac gtgtgatcag atatcgcggc cgctctagac 1380 caccatggga tggtcatgta tcatcctttt tctagtagca actgcaaccg gtgtacattc 1440 tgaggttaga ctgcgggagt caggaggggg gcttgtgaag cccggtgggt ctctccgcct 1500 gagctgttct gcctccggct ttgatttcga taacgcctgg atgacctggg tcaggcagcc 1560 tccaggtaag ggactggagt gggtgggaag aatcacaggt ccaggcgagg gctggtccgt 1620 ggactacgcg gaatctgtta aagggcggtt tacaatctca agggacaata ccaagaatac 1680 cttgtatttg gagatgaaca acgtgagaac tgaagacacc ggatattact tctgtgccag 1740 aacaggcaaa tactacgact tctggttcgg ctatccccct ggcgaggaat attttcaaga 1800 ctggggtcag ggaacccttg ttatcgtgtc ctccgcgtcg accaagggcc catcggtctt 1860 ccccctggca ccctcctcca agagcacctc tgggggcaca gcggccctgg gctgcctggt 1920 caaggactac ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc tgaccagcgg 1980 cgtgcacacc ttcccggctg tcctacagtc ctcaggactc tactccctca gcagcgtggt 2040 gaccgtgccc tccagcagct tgggcaccca gacctacatc tgcaacgtga atcacaagcc 2100 cagcaacacc aaggtggaca agaaagttga gcccaaatct tgtgacaaaa ctcacacatg 2160 cccaccgtgc ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa 2220 acccaaggac accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt 2280 gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa 2340 tgccaagaca aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct 2400 caccgtcctg caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa 2460 agccctccca gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc 2520 acaggtgtac accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac 2580 ctgcctggtc aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca 2640 gccggagaac aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct 2700 ctacagcaag ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc 2760 cgtgatgcat gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg 2820 taaatgatga ggatccagat ctgctgtgcc ttctagttgc cagccatctg ttgtttgccc 2880 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa 2940 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg 3000 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg 3060 ctctatgggt acccaggtgc tgaagaattg acccggttcc tcctgggcca gaaagaagca 3120 ggcacatccc cttctctgtg acacaccctg tccacgcccc tggttcttag ttccagcccc 3180 actcatagga cactcatagc tcaggagggc tccgccttca atcccacccg ctaaagtact 3240 tggagcggtc tctccctccc tcatcagccc accaaaccaa acctagcctc caagagtggg 3300 aagaaattaa agcaagatag gctattaagt gcagagggag agaaaatgcc tccaacatgt Page 72 3360

Sequence_Listing

gaggaagtaa tgagagaaat catagaattt taaggccatg atttaaggcc atcatggcct 3420 taatcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 3480 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 3540 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 3600 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 3660 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 3720 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 3780 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 3840 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 3900 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 3960 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 4020 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 4080 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 4140 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 4200 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 4260 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 4320 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 4380 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcggg 4440 gggggggggc gctgaggtct gcctcgtgaa gaaggtgttg ctgactcata ccaggcctga 4500 atcgccccat catccagcca gaaagtgagg gagccacggt tgatgagagc tttgttgtag 4560 gtggaccagt tggtgatttt gaacttttgc tttgccacgg aacggtctgc gttgtcggga 4620 agatgcgtga tctgatcctt caactcagca aaagttcgat ttattcaaca aagccgccgt 4680 cccgtcaagt cagcgtaatg ctctgccagt gttacaacca attaaccaat tctgattaga 4740 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat 4800 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga 4860 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta 4920 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat 4980 ccggtgagaa tggcaaaagc ttatgcattt ctttccagac ttgttcaaca ggccagccat 5040 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct 5100 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca 5160 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt 5220 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag 5280 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc 5340 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact Page 73 5400

Sequence_Listing

ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat 5460 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctcg 5520 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag 5580 cagacagttt tattgttcat gatgatatat ttttatcttg tgcaatgtaa catcagagat 5640 tttgagacac aacgtggctt tccccccccc cccattattg aagcatttat cagggttatt 5700 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 5760 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa 5820 cctataaaaa taggcgtatc acgaggccct ttcgtc 5856

<210> 82 <211> 461 <212> PRT <213> Artificial Sequence

<220> <223> Variant Antibody Sequence <400> 82 Glu Val Arg Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Asp Asn Ala 20 25 30 Trp Met Thr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Thr Gly Pro Gly Glu Gly Trp Ser Val Asp Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr 65 70 75 80 Leu Tyr Leu Glu Met Asn Asn Val Arg Thr Glu Asp Thr Gly Tyr Tyr 85 90 95 Phe Cys Ala Arg Thr Gly Lys Tyr Tyr Asp Phe Trp Phe Gly Tyr Pro 100 105 110 Pro Gly Glu Glu Tyr Phe Gln Asp Trp Gly Gln Gly Thr Leu Val Ile 115 120 125 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 130 135 140 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

145 150 155 160

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Lys Asp Tyr Phe Pro 165 Sequence_Listing Ala Glu Pro Val Thr Val 170 Ser Trp Asn Ser Gly 175 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 180 185 190 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 195 200 205 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 210 215 220 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 225 230 235 240 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 245 250 255 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 260 265 270 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 275 280 285 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 290 295 300 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 305 310 315 320 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 325 330 335 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 340 345 350 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 355 360 365 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 370 375 380 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 385 390 395 400 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 405 410 415 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 420 425 430

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Sequence_Listing

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 435 440 445

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 460 <210> 83 <211> 461 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 83

Glu 1 Val Arg Leu Arg 5 Glu Ser Gly Ser Leu Arg Leu 20 Ser Cys Ser Ala Trp Met Thr 35 Trp Val Arg Gln Pro 40 Gly Arg 50 Ile Thr Gly Pro Gly 55 Glu Ser 65 Val Lys Gly Arg Phe 70 Thr Ile Leu Tyr Leu Glu Met 85 Asn Asn Val Phe Cys Ala Arg 100 Thr Gly Lys Tyr Pro Gly Glu 115 Glu Tyr Phe Gln Asp 120 Val Ser 130 Ser Ala Ser Thr Lys 135 Gly Ser 145 Ser Lys Ser Thr Ser 150 Gly Gly Lys Asp Tyr Phe Pro 165 Glu Pro Val Leu Thr Ser Gly 180 Val His Thr Phe Leu Tyr Ser 195 Leu Ser Ser Val Val 200

Gly Gly 10 Leu Val Lys Pro Gly 15 Gly Ser 25 Gly Phe Asp Phe Asp 30 Asn Ala Pro Gly Lys Gly Leu 45 Glu Trp Val Gly Trp Ser Val 60 Asp Tyr Ala Glu Ser Arg Asp 75 Asn Thr Lys Asn Thr 80 Arg Thr 90 Glu Asp Thr Gly Tyr 95 Tyr Tyr 105 Asp Phe Trp Phe Gly 110 Tyr Pro Trp Gly Gln Gly Thr 125 Leu Val Ile Pro Ser Val Phe 140 Pro Leu Ala Pro Thr Ala Ala 155 Leu Gly Cys Leu Val 160 Thr Val 170 Ser Trp Asn Ser Gly 175 Ala Pro 185 Ala Val Leu Gln Ser 190 Ser Gly Thr Val Pro Ser Ser 205 Ser Leu Gly

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Sequence_Listing

Thr Gln Thr Tyr 210 Ile Cys Asn Val 215 Asn His Lys Pro 220 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 225 230 235 240 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 245 250 255 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 260 265 270 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 275 280 285 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 290 295 300 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 305 310 315 320 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 325 330 335 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 340 345 350 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 355 360 365 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 370 375 380 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 385 390 395 400 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 405 410 415 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 420 425 430 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 435 440 445 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 460

<210> 84 <211> 215

Page 77

Sequence_Listing <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 84

Ala Ser Glu 1 Leu Thr 5 Gln Asp Pro Ala Val 10 Ser Val Ala Leu Lys 15 Gln Thr Val Thr Ile Thr Cys Arg Gly Asp Ser Leu Arg Ser His Tyr Ala 20 25 30 Ser Trp Tyr Gln Lys Lys Pro Gly Gln Ala Pro Val Leu Leu Phe Tyr 35 40 45 Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ala Ser Gly Asn Arg Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Arg Asp Lys Ser Gly Ser Arg 85 90 95 Leu Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Gln Pro 100 105 110 Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu 115 120 125 Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro 130 135 140 Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala 145 150 155 160 Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala 165 170 175 Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg 180 185 190 Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr 195 200 205 Val Ala Pro Thr Glu Cys Ser

210 215 <210> 85 <211> 6 <212> PRT <213> Artificial Sequence

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Sequence_Listing <220>

<223> Variant Antibody Sequence <400> 85

Ser Leu Arg Cys His Trp 1 5 <210> 86 <211> 6 <212> PRT <213> Artificial Sequence <220>

<223> Variant Antibody Sequence <400> 86

Ser Leu Arg Cys His Phe 1 5

Page 79